Nucleic acids encoding immunotoxins containing a disulfide-stabilized antibody fragment replacing half or more of domain IB of pseudomonas exotoxin, and methods of use of the encoded immunotoxins

ABSTRACT

This invention provides for nucleic acids encoding immunotoxins comprising a Pseudomonas exotoxin (PE) that does not require proteolytic activation for cytotoxic activity attached to an Fv antibody fragment having a variable heavy chain region bound through at least one disulfide bond to a variable light chain region. The combination of a &#34;disulfide-stabilized&#34; binding agent fused to a PE that does not require proteolytic activation provides an immunotoxin having surprising cytotoxic activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. Ser. No. 08/809,668, filed Aug. 21, 1997,which is a national stage application of PCT/US96/16327, filed Oct. 11,1996, which is a continuation-in-part of U.S. Ser. No. 60/005,388, filedOct. 13, 1995. The contents of all of these applications are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

This invention pertains to the production and use of Pseudomonas-derivedimmunotoxins modified to increase their toxicity and potency in therapy.In particular, the immunotoxins of this invention include adisulfide-stabilized (ds) target-binding agent, such as the variableregion of an antibody molecule, and a Pseudomonas exotoxin that does notrequire proteolytic activation for cytotoxic activity.

Immunotoxins were initially produced by chemically coupling antibodiesto toxins (Vitetta et al. Cell, 41: 653-654 (1985); Pastan et al., Ann.Rev. Biochem. 61: 331-354 (1992)) to form chimeric molecules. In thesemolecules, the antibody portion mediated selective binding to targetcells, while the toxin portion mediated translocation into the cytosoland subsequent cell killing. Several toxins have been used to makeimmunotoxins including ricin A chain, blocked ricin, saporin, pokeweedantiviral protein, diphtheria toxin and Pseudomonas exotoxin A (PE)(Pastan et al., Science 254: 1173-1177 (1991); Vitetta et al., Semin.Cell Biol. 2: 47-58 (1991); Tazzari et al., Br. J. Hematol 81: 203-211(1992); Uckun et al., Blood, 79: 2201-2214 (1992)).

Several clinical trials with immunotoxins have shown activity againstlymphomas and other cancers derived from the hematopoietic system(Vitetta et al., Cancer Res. 51: 4052-4058 (1991); Grossbard et al., J.Clin. Oncol. 11: 726-737 (1993)). However, these immunotoxins areheterogeneous and their large size limits penetration into solid tumors.Second generation immunotoxins are totally recombinant molecules made byfusing the smallest functional module of an antibody, the Fv fragment,to a truncated toxin which lacks the cell-binding domain (Brinkmann etal., Proc. Natl. Acad. Sci. USA 88: 8616-8620 (1991); Kreitman et al.,Blood, 80: 2344-2352 (1992)). The small size of single-chainFv-immunotoxins makes them much more useful than chemical conjugates ofwhole antibodies for certain therapeutic applications because theirsmall size increases tumor penetration and efficacy (Fukimori et al,Cancer Res. 49: 5656-5663 (1989); Jain, Cancer Res., 50: 814-819 (1990);Sung et al., Cancer Res. 50: 7382-7392 (1990)).

Several types of recombinant Fv-immunotoxins containing PE have beenmade and tested in vitro as well as in animal models (Brinkmann et al.,Proc. Natl. Acad. Sci. USA 88: 8616-8620 (1991); Kreitman et al., Blood,80: 2344-2352 (1992); Batra et al., Proc. Natl. Acad. Sci. USA 89:5867-5871 (1992); Reiter et al., Cancer Res. 54: 2714-2718 (1994);Brinkmann et al, Proc. Natl. Acad. Sci. USA 90: 547-551 (1993)).Initially, the Fv regions of the immunotoxins were arranged in asingle-chain form (scFv-immunotoxin) with the V_(H) and V_(L) domainsconnected by a linking peptide. More recently, disulfide-stabilizedforms of Fv-immunotoxins (dsFv-immunotoxins) have been generated inwhich the V_(H) and V_(L) domains are connected by a disulfide bondengineered into the framework region (see, e.g. copending applicationU.S. Ser. No. 08/077,252 filed on Jun. 14, 1993; Brinkmann et al., Proc.Natl. Acad. Sci. USA 90: 7538-7542 (1993); Reiter et al., Protein Eng.,7: 697-704 (1994)). Disulfide-stabilized Fv immunotoxins are much morestable than single-chain immunotoxins and can show improved binding toantigen (Reiter et al., J. Biol. Chem. 269: 18327-18331 (1994); Reiteret al., Protein Eng. 7: 697-704 (1994)). In addition, dsfv-immunotoxinsare slightly smaller in size than scfv-immunotoxins, and may exhibitbetter tumor penetration.

Recombinant immunotoxins containing PE must be proteolytically activatedwithin the cell by cleavage in domain f between amino acids 279 and 280(Ogata et al J. Biol Chem., 267: 25369-25401 (1992)). To eliminate theneed for intracellular proteolytic activation and thereby increasecytotoxic activity, the toxin moiety of recombinant toxins has beenmodified. This was initially done with recombinant toxins containingTGFα by producing a truncated toxin (PE280-613) with TGFα inserted nearthe end of domain III at position 607 (Theuer et al, J. Urol., 149:1626-1632 (1993); Theuer et al., Cancer Res., 53: 340-347 (1993)).Because the toxin begins at position 280, it does not need proteolyticactivation within the cell (Ogata et al. J. Biol. Chem., 267:25369-25401 (1992); Theuer et al. J. Biol. Chem., 267: 16872-16877(1992)). In addition, these molecules had two other mutations. One was adeletion of unnecessary residues in domain Ib (365-380). The other wasto change the carboxyl terminus from REDLK (SEQ ID NO:8) to KDEL (SEQ IDNO:9) to increase cytotoxic activity (Seetharam et al. J. Biol. Chem.,266: 17376-17381 (1991)). This molecule termed PE35/TGFαKDEL was 10-700fold more active than TGFα-PE40 on several human bladder cancer celllines (Theuer et al, J. Urol., 149: 1626-1632 (1993)). However, evenmore specific and reactive immunotoxins are desired.

SUMMARY OF THE INVENTION

The present invention is premised, in part, on the discovery thatimmunotoxins comprising both a disulfide-stabilized binding agent and aPseudomonas exotoxin modified so that proteolytic cleavage is notrequired for cytotoxicity, show cytotoxicity far greater than would beexpected based on the performance of fusion proteins comprising eitherthe disulfide stabilized binding protein or the modified Pseudomonasexotoxin alone.

Thus, in one embodiment, this invention provides for an immunotoxincomprising a Pseudomonas exotoxin (PE) that does not require proteolyticactivation for cytotoxic activity attached to a variable heavy (V_(H))region of an Fv antibody fragment where the variable heavy region isbound through at least one disulfide bond to a variable light (V_(L))chain region. In a preferred embodiment, the Pseudomonas exotoxin is atruncated Pseudomonas exotoxin lacking domain Ia. In another embodiment,Pseudomonas exotoxin lacks residues 1 through 279. The variable heavychain region can substantially replace domain Ib of the Pseudomonasexotoxin, or alternatively, it can be located in the carboxyl terminusof the Pseudomonas exotoxin. The amino terminus of the heavy chainregion can be attached to the PE through a peptide linker (e.g. SGGGGS;SEQ ID NO:10). The carboxyl terminus of the heavy chain region can alsobe attached to the PE through a peptide linker (e.g., KASGGPE; SEQ IDNO:11). In a preferred embodiment, the antibody fragment is from B1, B3,B5, e23, BR96, anti-Tac, RFB4, or HB21, more preferably from B1, B3, B5,and e23. The carboxyl terminal sequence of the immunotoxin can be KDEL(SEQ ID NO:9). Particularly preferred immunotoxins includePE35/e23(dsfv)KDEL and B1 (dsFv)PE33.

In another embodiment, the variable light (V_(L)) region rather than thevariable heavy region (V_(H)) is attached (fused) to the Pseudomonasexotoxin, while the variable heavy (V_(H)) chain is bound to thevariable light (V_(L)) chain through at least one disulfide bond.Particularly preferred embodiments include all of the embodimentsdescribed above differing only in that the V_(L) chain is substitutedfor the V_(H) chain and vice versa.

This invention also provides for nucleic acids encoding all of theabove-described immunotoxins. Thus, in one embodiment, this inventionprovides for a nucleic acid encoding an immunotoxin comprising a heavychain variable region of an Fv antibody fragment attached to aPseudomonas exotoxin that does not require proteolytic activation forcytotoxic activity. The encoded heavy chain variable region containscysteine residues that form disulfide linkages with a variable lightchain region of an Fv fragment and the antibody fragments comprise thevariable light or variable heavy chains of B1, B3, B5, e23, BR96,anti-Tac, RFB4, or HB21. In a preferred embodiment, the nucleic acidencodes an immunotoxin in which the heavy chain variable region issubstituted for domain Ib of the Pseudomonas exotoxin. In anotherembodiment, the nucleic acid encodes an immunotoxin in which the heavychain variable region is located after residue 607 of the Pseudomonasexotoxin. The PE component of the encoded immunotoxin preferably lacksamino acid residues 1 through 279. In another preferred embodiment, thisinvention also provides for nucleic acids as described above encodingimmunotoxins in which the V_(L) chain is substituted for the V_(H) chainand vice versa.

It was also a discovery of this invention that single chain immunotoxinscomprising V_(L) or V_(H) regions alone, rather than as components of Fvfragments, are capable of binding their target molecules. Thus, in yetanother embodiment, this invention provides for a single chainimmunotoxin fusion protein comprising a Pseudomonas exotoxin (PE) thatdoes not require proteolytic activation for cytotoxic activity attachedto a variable light (V_(L)) or a variable heavy (V_(H)) chain region.Suitable toxin components include any of the Pseudomonas exotoxinsdescribed above. In a preferred embodiment, the Pseudomonas exotoxin isa truncated Pseudomonas exotoxin lacking domain Ia. In another preferredembodiment, the Pseudomonas exotoxin lacks residues 1 through 279. Thevariable heavy or light chain can substantially replace domain Ib, orcan be located in the carboxyl terminus of the Pseudomonas exotoxin. Theamino terminus of the variable heavy or light chain region can beattached to the PE through a peptide linker (e.g., SGGGGS) while thecarboxyl terminus of the variable heavy or light chain region can beattached to the PE through a peptide linker (e.g., KASGGPE). Thevariable heavy or light chain are preferably derived from B1, B3, B5,e23, BR96, anti-Tac, RFB4, or HB21, and more preferably from Bi, B3, B5and e23. The immunotoxin can have the carboxyl terminal sequence KDEL.

In another embodiment, this invention provides for nucleic acidsencoding any of the above-described single chain immunotoxin fusionproteins.

This invention also provides for methods of killing cells bearing acharacteristic marker. The methods comprise contacting the cells withany of the above-described immunotoxins comprising a Pseudomonasexotoxin (PE) that does not require proteolytic activation for cytotoxicactivity attached to a heavy chain region of an Fv antibody fragmentwhich is bound through at least one disulfide bond to a variable lightchain region or, conversely, attached to a light chain region of an Fvantibody fragment which is bound through at least one disulfide bond toa variable heavy chain region.

The immunotoxins of this invention are suitable for use inpharmacological compositions. This invention thus provides for apharmaceutical composition comprising an effective amount of animmunotoxin in a pharmacologically acceptable excipient. Preferredimmunotoxins include any of the above-described immunotoxins comprisinga Pseudomonas exotoxin (PE) that does not require proteolytic activationfor cytotoxic activity attached to a heavy chain region of an Fvantibody fragment which is bound through at least one disulfide bond toa variable light chain region or, conversely, attached to a light chainregion of an Fv antibody fragment which is bound through at least onedisulfide bond to a variable heavy chain region.

Finally, this invention also provides methods of delivering an antibodyto the cytosol of a cell. The methods involve contacting the cell with achimeric molecule comprising the antibody attached to a Pseudomonasexotoxin that does not require proteolytic cleavage for translocationinto the cytosol of said cell. The chimeric molecule is preferably afusion protein in which the antibody (e.g., a V_(H) or a V_(L) region)is substituted into domain Ib, domain II or the carboxyl terminus ofdomain III. Domain III is preferably inactivated (its cytotoxic activitysubstantially eliminated) by truncation, mutation, or insertion of aheterologous peptide sequence.

DEFINITIONS

Abbreviations for the twenty naturally occurring amino acids followconventional usage (Immunology--A Synthesis, (2nd ed., E. S. Golub andD. R. Gren, eds., Sinauer Associates, Sunderland, Mass., 1991).Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α,α-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for polypeptides of the presentinvention. Examples of unconventional amino acids include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, ω-N-methylarginine, and othersimilar amino acids and imino acids (e.g., 4-hydroxyproline). In thepolypeptide notation used herein, the left-hand direction is theamino-terminal direction and the right-hand direction is thecarboxy-terminal direction, in accordance with standard usage andconvention. Similarly, unless specified otherwise, the left hand end ofsingle-stranded polynucleotide sequences is the 5' end; the left handdirection of double-stranded polynucleotide sequences is referred to asthe 5' direction. The direction of 5' to 3' addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5' to the 5' end of the RNA transcript are referred to as "upstreamsequences"; sequence regions on the DNA strand having the same sequenceas the RNA and which are 3' to the 3' end of the RNA transcript arereferred to as "downstream sequences".

The term "nucleic acid" refers to a single or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5' to the 3'end. It includes self-replicating plasmids, infectious polymers of DNAor RNA and non-functional DNA or RNA.

The phrase "specifically binds to a protein" or "specificallyimmunoreactive with", when referring to an antibody or a "binding agent"refers to a binding reaction which is determinative of the presence ofthe target molecule (e.g. protein) in the presence of a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified binding agents or fusion proteinscomprising the specified binding agents bind to a particular protein, orother target molecule, and do not bind in a significant amount to otherproteins present in the sample. Specific binding to a protein under suchconditions may require a binding agent that is selected for itsspecificity for a particular target molecule. For example, antibodiesB1, B3, B5 and BR96 bind the Lewis^(Y) carbohydrate antigen and not toany other target molecules present in a biological sample. A variety ofimmunoassay formats may be used to select binding agents specificallyreactive with a particular target molecule. For example, solid-phaseELISA immunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with a protein. See Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity.

"Peptides" and "polypeptides" are chains of amino acids whose α carbonsare linked through peptide bonds formed by a condensation reactionbetween the a carbon carboxyl group of one amino acid and the aminogroup of another amino acid. The terminal amino acid at one end of thechain (amino terminal) therefore has a free amino group, while theterminal amino acid at the other end of the chain (carboxy terminal) hasa free carboxyl group.

Typically, amino acids comprising a polypeptide are numbered in order,increasing from the amino terminal to the carboxy terminal of thepolypeptide. Thus when one amino acid is said to "follow" another, thatamino acid is positioned closer to the carboxy terminal of thepolypeptide then the "preceding" amino acid.

The term "residue" as used herein refers to an amino acid that isincorporated into a peptide. The amino acid may be a naturally occurringamino acid and, unless otherwise limited, may encompass known analogs ofnatural amino acids that can function in a similar manner as naturallyoccurring amino acids.

The term "domain" refers to a characteristic region of a polypeptide.The domain may be characterized by a particular structural feature suchas an alpha helix, or a β pleated sheet, by characteristic constituentamino acids (e.g. Predominantly hydrophobic or hydrophilic amino acids,or repeating amino acid sequences), or by its localization in aparticular region of the folded three dimensional polypeptide. A domainmay be composed of a series of contiguous amino acids or by amino acidsequences separated from each other in the chain, but brought intoproximity by the folding of the polypeptide.

A "fusion protein" refers to a polypeptide formed by the joining of twoor more polypeptides through a peptide bond formed between the aminoterminus of one polypeptide and the carboxyl terminus of anotherpolypeptide. The fusion protein may be formed by the chemical couplingof the constituent polypeptides or it may be expressed as a singlepolypeptide from nucleic acid sequence encoding the single contiguousfusion protein. A single chain fusion protein is a fusion protein havinga single contiguous polypeptide backbone.

A "spacer" or "linker" as used herein refers to a peptide that joins theproteins comprising a fusion protein. Generally a spacer has no specificbiological activity other than to join the proteins or to preserve someminimum distance or other spatial relationship between them. However,the constituent amino acids of a spacer may be selected to influencesome property of the molecule such as the folding, net charge, orhydrophobicity of the molecule.

A "target molecule", as used herein, refers to a molecule to which thebinding agent specifically binds. Typically target molecules arecharacteristic of a particular cell type or physiological state. Thus,for example, target molecules such as Lewis^(Y) antigen or c-erbB2 aretypically found on various cancer cells. Binding agents directed tothese target molecules thus direct the immunotoxins to the cells bearingthe target molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic representation of B1 immunotoxins having adisulfide-stabilized binding agent placed at the amino terminus orinserted in place of domain Ib. Positions of amino acids that span PEsequences are numbered. The arrow sign marks the proteolytic site of PEfor activation. S--S shows the disulfide bond linkage between the Fvfragments. L: peptide linker; V_(H) : variable heavy chain; V_(L) :variable light chain; II: PE domain II for translocation; Ib: PE domainIb (function unknown); III: PE domain III for ADP-ribosylation of EF2.

FIG. 2 provides a schematic representation of e23 immunotoxins having acarboxyl disulfide-stabilized binding agent. Position of the amino acidsthat span PE sequences are numbered. The amino acids listed in theone-letter code are the C-terminal residues (KDEL=SEQ ID NO:9). Thearrow sign marks the proteolytic site of PE for activation. S--S showsthe disulfide bond linkage between the Fv fragments Linker SGGGGS=SEQ IDNO:10.

FIG. 3 illustrates the plasmids used for expression of e23 dsfvimmunotoxins. Positions of cysteine replacement (shown as asterisk star)in framework region of e23(Fv) are Asn⁴⁴ →Cys in V_(H) and Gly⁹⁹ →Cys inV_(L). Plasmid pCT12 encodes a protein termed PE35/TGFαKDEL, startingwith a Met at position 280 of PE and containing amino acids 281 to 364and 381 to 607 with a gene encoding TGFα inserted between amino acid 607and 604 of PE, and the carboxyl-terminal amino acids KDEL (SEQ ID NO:9)are substituted for the native REDLK sequence (SEQ ID NO:8). PlasmidpCTK101 and pCTK103, encoding PE35/e23(V_(H) Cys₄₄)KDEL andPE35/e23(V_(H) Cys₄₄), respectively, are the expression plasmid for thetoxin-V_(H) components of the dsfv immunotoxin PE/e23(dsfv)KDEL(SGGGGS=SEQ ID NO:10). Plasmid pCTK102 encodes e23(V_(L)) Cys 99 fusedto PE amino acids 604-608 and carboxyl terminal sequences KDEL (SEQ IDNO:9). Plasmids pYR39 and pYR40 encode e23(V_(H) Cys₄₄)PE38KDEL ande23(V_(L) Cys₉₉), respectively.

FIG. 4 shows the anti-tumor effect and durability of complete remissionscaused by B1 (dsfv)PE33 and B1(dsFv)PE38 in a nude mouse model. Group offive mice were injected s.c. with 3×10⁶ on day 0 and were treated byi.v. injections of (A) B1(dsFv)PE33 or (B) B1(dsFv)PE38 on days 5, 7,and 9 (indicated by vertical arrows) when the tumors were established.Control mice were treated with PBS-HSA. Error bars represent thestandard error of the data. (∘) Control; (□) 400 pmole/kg; (▴) 200pmole/kg; (Δ) 100 pmole/kg.

FIG. 5 provides a schematic representation of B3 immunotoxins having adisulfide-stabilized binding agent placed at the carboxy terminus orinserted in place of domain Ib. Positions of amino acids that span PEsequences are numbered. S--S shows the disulfide bond linkage betweenthe Fv fragments. V_(H) : variable heavy chain; V_(L) : variable lightchain; II: PE domain II for translocation; III: PE domain III forADP-ribosylation of EF2.

DETAILED DESCRIPTION

This invention relates to Pseudomonas exotoxin (PE) based immunotoxinshaving increased cytotoxic activity. It was a surprising discovery ofthe present invention that immunotoxins comprising adisulfide-stabilized binding agent attached to a Pseudomonas exotoxinthat has been modified so that proteolytic cleavage is not required forcytotoxic activity show unexpected high levels of cytotoxicity,particularly greater than a ten-fold increase in cytotoxicity to targetcells. This cytotoxicity combined with the smaller size of theimmunotoxin which provides greater penetration into solid tumors resultsin an immunotoxin of improved pharmacological efficacy. The term bindingagent, as used herein, refers to a molecule that specifically recognizesand binds to a particular preselected target molecule. The binding agentis thus capable of specifically targeting cells that express preselectedtarget molecule. Thus chimeric immunotoxins including a binding agentspecifically bind to and kill or inhibit growth of cells bearing targetmolecules recognized by the binding agent.

Preferred binding agents are immunoglobulins, members of theimmunoglobulin family or molecules derived from immunoglobulins ormembers of the immunoglobulin family as described below in SectionII(A). Particularly preferred binding agents include immunoglobulinfragments incorporating recognition domains of the immunoglobulin (orimmunoglobulin family) molecules (e.g. incorporating the variable regionof an antibody).

Preferred binding agents include at least two different polypeptidesthat are joined together by a linker, most preferably by a disulfidelinkage (e.g. formed between respective cysteines in each chain).Binding agents comprising two polypeptide chains joined by a disulfidelinkage have a reduced tendency to aggregate, show a generally longerserum half-life and are said to be "stabilized". Thus adisulfide-stabilized binding agent, as used herein, refers to a bindingagent comprising at least two polypeptides joined by at least onedisulfide linkage. The disulfide linkage, however, need not be the onlylinkage joining the polypeptides. Thus, for example, a variable lightand variable heavy chain of an antibody may be joined by a disulfidelinkage and additionally joined by terminal peptide linker. Such amolecule may thus be expressed as a single chain fusion protein (e.g.V_(H) -peptide-V_(L)) where the V_(H) and V_(L) polypeptides aresubsequently cross-inked by the formation of a disulfide linkage.Methods of producing disulfide-stabilized binding agents can be found incopending patent application U.S. Ser. No. 08/077,252, filed on Jun. 14,1993.

As indicated above, the disulfide-stabilized binding agent is attachedto a Pseudomonas exotoxin which is modified so that it is cytotoxicwithout requiring proteolytic activation. As explained below in SectionIII, this typically entails truncating the amino terminus to at leastposition 279. Methods of producing Pseudomonas exotoxins that do notrequire proteolytic cleavage for activation are described in copendingpatent application Ser. No. 08/405,615, filed on Mar. 15, 1995 which isa continuation of Ser. No. 07/901,709 filed on Jun. 18, 1992.

The disulfide-stabilized binding agent may be located at virtually anyposition within the modified Pseudomonas exotoxin. In one preferredembodiment, the binding agent is inserted in replacement for domain Iaas has been accomplished in what is known as the TGFα/PE40 molecule(also referred to as TP40) described in Heimbrook et al., Proc. Natl.Acad. Sci., USA, 87: 4697-4701 (1990) and in commonly assigned U.S. Ser.No. 07/865,722 filed Apr. 8, 1992 and in U.S. Ser. No. 07/522,563 filedMay 14, 1990.

The disulfide-stabilized binding agent may additionally substitute forall of domain Ib or portions of it. Thus, for example residues 343through 394 in domain Ib may be eliminated or replaced with one of thetwo chains of the disulfide-stabilized binding agent.

The disulfide-stabilized binding agent may alternatively be located nearor at the amino or carboxyl terminus. Where the disulfide-stabilizedbinding agent is located in the carboxyl terminus, it is preferablylocated after amino acid 604, with a position between amino acid 604 and608 being more preferred and a position after about amino acid 607 beingmost preferred. An appropriate carboxyl end of PE can be recreated byplacing amino acids about 604-613 of PE after the binding agent. Thus,the disulfide-stabilized binding agent is preferably inserted within therecombinant PE molecule after about amino acid 607 and is followed byamino acids 604-613 of domain III of PE. The new carboxyl terminus canalso include the endoplasmic retention sequences REDLK (SEQ ID NO:8) andKDEL (SEQ ID NO:9), with KDEL (SEQ ID NO:9) being most preferred. Theterminus may also include terminal PE amino acids. Thus, in oneparticularly preferred embodiment, the disulfide-stabilized bindingagent is an antibody which is located after residue 607 and thenfollowed by PE residues 604-608 which, in turn, are followed by KDEL(SEQ ID NO:9). The V_(L) or V_(H) regions from a desired antibody mayalso be inserted in a single chain form within domain III.

Where the disulfide-stabilized binding agent is an antibody, moreparticularly a Fv region of an antibody, the modified PE can be fused toeither the V_(H) or the V_(L) domain of the Fv in any of the PE regionsas described above. The fusion between the PE and the V_(L) or V_(H) canbe direct or through one or more peptide linker(s). Such linkers can beattached to the V_(H) or the V_(L) at either the carboxyl terminal ofthe variable chain, the amino terminal of the variable chain, or at bothtermini.

When the variable heavy (V_(H)) chain is fused to the PE, the variablelight (V_(L)) chain is joined to the fused variable heavy chain by oneor more disulfide linkages. Conversely, when the variable light (V_(L))chain is fused to the PE, the variable heavy (V_(H)) chain is joined tothe fused variable light chain by one or more disulfide linkages.

It was also a discovery of the present invention that variable heavy orlight chain regions alone, rather than as a component of an Fv region,are capable of specifically binding to their target molecules. Thus, inone embodiment, this invention provides for single chain immunotoxinfusion proteins comprising a Pseudomonas exotoxin (PE) that does notrequire proteolytic activation for cytotoxic activity attached to avariable light (V_(L)) or a variable heavy (V_(H)) chain region. Ineffect these fusion proteins are made in the same manner as thedisulfide-stabilized fusion proteins described above, but the stepwhereby the respective variable regions are joined by disulfide linkagesis omitted. In addition, as no disulfide linkages need be formed, thereis no need to introduce cysteine into either of the variable regions, orto eliminate cysteines existing in the PE. Either the variable lightchain or the variable heavy chain can be expressed in fusion with themodified PE.

Those skilled in the art will realize that additional modifications,deletions, insertions and the like may be made to thedisulfide-stabilized binding agent and PE genes. Especially, deletionsor changes may be made in PE or in a linker connecting an antibody geneto PE, in order to increase cytotoxicity of the fusion protein towardtarget cells or to decrease nonspecific cytotoxicity toward cellswithout antigen for the antibody. Typical modifications, include, butare not limited to introduction of an upstream methionine fortranscription initiation, mutation of residues to cysteine in the V_(H)or V_(L) regions for the creation of disulfide linkages, mutation ofcysteine at position 287 in PE to serine to prevent unwanted disulfidelinkage formation, an upstream (amino) peptide linker (e.g. GGGGS; SEQID NO:12), a downstream (carboxyl) peptide linker (e.g. KASGGPE; SEQ IDNO:11), and so forth. All such constructions may be made by methods ofgenetic engineering well known to those skilled in the art (see,generally, Berger and Kimmel, Guide to Molecular Cloning Techniques,Methods in Enzymology Volume 152 Academic Press, Inc., San Diego, Calif.(Berger); Sambrook et al. Molecular Cloning--A Laboratory Manual (2nded.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press,NY, (1989); and Current Protocols in Molecular Biology, F. M. Ausubel etal., eds., a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel). Methods ofproducing recombinant immunoglobulins are also known in the art. See,Cabilly, U.S. Pat. No. 4,816,567; and Queen et al. Proc. Nat'l Acad.Sci. USA, 86: 10029-10033 (1989)).

I. Disulfide Stabilized Binding Protein

A) General immunoglobulin structure.

As used herein, the terms "immunological binding" and "immunologicalbinding properties" refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smaller Kdrepresents a greater affinity. Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and on geometric parameters that equally influence therate in both directions. Thus, both the "on rate constant" (K_(on)) andthe "off rate constant" (K_(off)) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation.The ratio of K_(off) /K_(on) enables cancellation of all parameters notrelated to affinity and is thus equal to the dissociation constantK_(d). (See, generally, Davies et al. Ann. Rev. Biochem., 59: 439-473(1990)).

As used herein, an "antibody" refers to a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as myriad immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one "light" (about 25 kD or about214 amino acids) and one "heavy" chain (about 50-70 kD or about 446amino acids). The C-terminus of each chain defines a constant region (C)that determines the antibody's effector function (e.g., complementfixation, opsonization, etc.), while the N-terminus of each chaindefines a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The terms variable lightchain (V_(L) ) and variable heavy chain (V_(H)) refer to these light andheavy chains respectively.

Members of the immunoglobulin family all share an immunoglobulin-likedomain characterized by a centrally placed disulfide bridge thatstabilizes a series of antiparallel β strands into animmunoglobulin-like fold. Members of the family (e.g., MHC class I,class II molecules, antibodies and T cell receptors) can share homologywith either immunoglobulin variable or constant domains.

Full-length immunoglobulin or antibody "light chains" (generally about25 kilodaltons (Kd), about 214 amino acids) are encoded by a variableregion gene at the N-terminus (generally about 110 amino acids) and aconstant region gene at the COOH-terminus. Full-length immunoglobulin orantibody "heavy chains" (generally about 50 Kd, about 446 amino acids),are similarly encoded by a variable region gene (generally encodingabout 116 amino acids) and one of the constant region genes (encodingabout 330 amino acids). Typically, the "V_(L) " will include the portionof the light chain encoded by the V_(L) and J_(L) (J or joining region)gene segments, and the "V_(H) " will include the portion of the heavychain encoded by the V_(H), and DH (D or diversity region) and J_(H)gene segments. See generally, Roitt, et al., Immunology, Chapter 6, (2ded. 1989) and Paul, Fundamental Immunology; Raven Press (2d ed. 1989).The Fv antibody fragment includes the variable heavy chain and variablelight chain regions.

An immunoglobulin light or heavy chain variable region comprises threehypervariable regions, also called complementarity determining regionsor CDRs, flanked by four relatively conserved framework regions or FRs.Numerous framework regions and CDRs have been described (see, Kabat etal., Sequences of Proteins of Immunological Interest, U.S. GovernmentPrinting Office, NIH Publication No. 91-3242 (1991); referred to hereinas "Kabat and Wu"). The sequences of the framework regions of differentlight or heavy chains are relatively conserved. The CDR and FRpolypeptide segments are designated empirically based on sequenceanalysis of the Fv region of preexisting antibodies or of the DNAencoding them. From alignment of antibody sequences of interest withthose published in Kabat and Wu and elsewhere, framework regions andCDRs can be determined for the antibody or other ligand binding moietyof interest. The combined framework regions of the constituent light andheavy chains serve to position and align the CDRs. The CDRs areprimarily responsible for binding to an epitope of an antigen and aretypically referred to as CDR1, CDR2, and CDR3, numbered sequentiallystarting from the N-terminus of the variable region chain. Frameworkregions are similarly numbered.

The general arrangement of T cell receptor genes is similar to that ofantibody heavy chains, T cell receptors (TCR) have both variable domains(V) and constant (C) domains. The V domains function to bind antigen.There are regions in the V domain homologous to the framework CDRregions of antibodies. Homology to the immunoglobulin V regions can bedetermined by alignment. The V region of the TCRs has a high amino acidsequence homology with the Fv of antibodies. Hedrick et al, Nature(London) 308:153-158 (1984)).

The term CDR, as used herein, refers to amino acid sequences whichtogether define the binding affinity and specificity of the naturalvariable binding region of a native immunoglobulin binding site (such asFv), a T cell receptor (such as V.sub.α and V.sub.β), or a syntheticpolypeptide which mimics this function. The term "framework region" or"FR", as used herein, refers to amino acid sequences interposed betweenCDRs.

The "binding agents" referred to here are those molecules that have avariable domain that is capable of functioning to bind specifically orotherwise recognize a particular ligand or antigen. Moieties ofparticular interest include antibodies and T cell receptors, as well assynthetic or recombinant binding fragments of those such as Fv, Fab,F(ab')₂ and the like. Appropriate variable regions include V_(H), V_(L),V.sub.α, and V.sub.β and the like.

Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)'₂, a dimer of Fab whichitself is a light chain joined to V_(H) -C_(H) 1 by a disulfide bond.The F(ab)'₂ may be reduced under mild conditions to braak the disulfidelinkage in the hinge region thereby converting the (Fab')₂ dimer into anFab' monomer. The Fab' monomer is essentially an Fab with part of thehinge region. The Fv region is the variable part of Fab; a V_(H) -V_(L)dimer (see, Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y.(1993), for a more detailed description of other antibody fragments).While various antibody fragments are defined in terms of the digestionof an intact antibody, one of skill will appreciate that such fragments(e.g., Fv) may be synthesized de novo either chemically or by utilizingrecombinant DNA methodology. Thus, the term antibody, as used hereinalso includes antibody fragments either produced by the modification ofwhole antibodies or synthesized de novo using recombinant DNAmethodologies. Preferred antibodies include disulfide-stabilizedantibodies, more preferably disulfide-stabilized Fv (dsfv) antibodies inwhich a variable heavy and a variable light chain are joined together byat least one disulfide linkage to form an intact Fv fragment.

Practice of this invention preferably employs the Fv portions of anantibody or the V portions of a TCR. Other sections, e.g., C_(H) andC_(L), of native immunoglobulin protein structure need not be presentand normally are intentionally omitted from the polypeptides of thisinvention. However, the polypeptides of the invention may compriseadditional polypeptide regions defining a bioactive region, e.g., atoxin or enzyme, or a site onto which a toxin or a remotely detectablesubstance can be attached, as will be described below.

B) Preparation of Fv Fragments

Information regarding the Fv antibody fragments or other ligand bindingmoiety of interest is required in order to produce proper placement ofthe disulfide bond to stabilize the desired disulfide stabilizedfragment, such as an Fv fragment (dsFv). The amino acid sequences of thevariable fragments that are of interest are compared by alignment withthose analogous sequences in the well-known publication by Kabat and Wu,supra, to determine which sequences can be mutated so that cysteine isencoded for in the proper position of each heavy and light chainvariable region to provide a disulfide bond in the framework regions ofthe desired polypeptide fragment. Cysteine residues are preferred toprovide the covalent disulfide bonds. For example, a disulfide bondcould be placed to connect FR4 of V_(L) and FR2 of V_(H) ; or to connectFR2 of V_(L) and FR4 of V_(H).

After the sequences are aligned, the amino acid positions in thesequence of interest that align with the following positions in thenumbering system used by Kabat and Wu are identified: positions 43, 44,45, 46, and 47 (group 1) and positions 103, 104, 105, and 106 (group 2)of the heavy chain variable region; and positions 42, 43, 44, 45, and 46(group 3) and positions 98, 99, 100, and 101 (group 4) of the lightchain variable region. In some cases, some of these-positions may bemissing, representing a gap in the alignment.

Then, the nucleic acid sequences encoding the amino acids at two ofthese identified positions are changed such that these two amino acidsare mutated to cysteine residues. The pair of amino acids to be selectedare, in order of decreasing preference:

V_(H) 44-V_(L) 105

V_(H) 44-V_(L) 99

V_(H) 44-V_(L) 100,

V_(H) 105-V_(L) 43,

V_(H) 105-V_(L) 42,

V_(H) 44-V_(L) 101,

V_(H) 10-V_(L) 43,

V_(H) 104-V_(L) 43,

V_(H) 45-V_(L) 98,

V_(H) 46-V_(L) 98,

V_(H) 103-V_(L) 43,

V_(H) 103-V_(L) 44,

V_(H) 103-V_(L) 45.

Most preferably, substitutions of cysteine are made at the positions:

V_(H) 44-V_(L) 105 (see, e.g., B1(dsfv)-PE33);

V_(H) 44-V_(L) 99 (see, e.g., PE35/e23(dsFv)KDEL);

V_(H) 44-V_(L) 100; or

V_(H) 105-V_(L) 43.

(The notation V_(H) 44-V_(L) 100, for example, refers to a polypeptidewith a V_(H) having a cysteine at position 44 and a cysteine in V_(L) atposition 100; the positions being in accordance with the numbering givenby Kabat and Wu.)

Note that with the assignment of positions according to Kabat and Wu,the numbering of positions refers to defined conserved residues and notto actual amino acid positions in a given antibody. For example, CysL100(of Kabat and Wu) which is used to generate ds(Fv)B3 as described in theexample below, actually corresponds to position 105 of B3(V_(L)).

In the case of V.sub.α and V.sub.β of T cell receptors, reference canalso be made to the numbering scheme in Kabat and Wu for T cellreceptors. Substitutions of cysteines can be made at position 41, 42,43, 44 or 45 of V.sub.α and at position 106, 107, 108, 109 or 110 ofV.sub.β ; or at position 104, 105, 106, 107, 108 or 109 of V.sub.α andat position 41, 42, 43, 44 or 45 of V.sub.β, such positions being inaccordance with the Kabat and Wu numbering scheme for TCRs. When suchreference is made, the most preferred cysteine substitutions are V.sub.α42-V.sub.β 110 and V.sub.α 108-V.sub.β 42. V.sub.α positions 106, 107and V.sub.β positions 104, 105 are CDR positions, but they are positionsin which disulfide bonds can be stably located.

As an alternative to identifying the amino acid position for cysteinesubstitution with reference to the Kabat and Wu numbering scheme, onecould align a sequence of interest with the sequence for monoclonalantibody (MAb) B1, B3, or B5 hybridomas of which have all been depositedwith the American Type Culture Collection in Rockville, Md. withdesignations of HB 10569, HB 10572, and HB 10573) as described in U.S.Pat. No. 5,242,813, copending application U.S. Ser. No. 07/767,331 filedon Sep. 30, 1991, copending application U.S. Ser. No. 08/051,133, filedon Apr. 22, 1993, copending applications U.S. Ser. No. 08/331,391,08/331,397 and 08/331,396, all filed on Oct. 28, 1994, and by Benhar etal., Clin. Cancer. Res., 1: 1023-1029 (1995). The amino acid positionsof B3 which correlate with the Kabat and Wu V_(H) positions set forthabove for Group 1 are 43, 44, 45, 46, and 47, respectively; for Group 2are 109, 110, 111, and 112, respectively. The amino acid positions of B3which correlate with the Kabat and Wu V_(L) positions set forth abovefor Group 3 are 47, 48, 49, 50 and 51, respectively; Group 4 are 103,104, 105, and 106, respectively.

Alternatively, the sites of mutation to the cysteine residues can beidentified by review of either the actual antibody or the model antibodyof interest as exemplified below. Computer programs to create models ofproteins such as antibodies are generally available and well-known tothose skilled in the art (see Kabat and Wu; Loew, et al., Int. J. Quant.Chem., Quant. Biol Symp., 15:55-66 (1988); Bruccoleri, et al., Nature,335:564-568 (1988); and Chothia, et al., Science, 233:755-758 (1986)).Commercially available computer programs can be used to display thesemodels on a computer monitor, to calculate the distance between atoms,and to estimate the likelihood of different amino acids interacting(see, Ferrin, et al., J. Mol. Graphics, 6: 13-27 (1988)). For example,computer models can predict charged amino acid residues that areaccessible and relevant in binding and then conformationaly restrictedorganic molecules can be synthesized. See, for example, Saragovi, etal., Science, 253:792 (1991). In other cases, an experimentallydetermined actual structure of the antibody may be available.

A pair of suitable amino acid residues should (1) have a C.sub.α-C.sub.α distance between the two residues less than or equal to 8 Å,preferably less than or equal to 6.5 Å (determined from the crystalstructure of antibodies which are available such as those from theBrookhaven Protein Data Bank) and (2) be as far away from the CDR regionas possible. Once they are identified, they can be substituted withcysteines. The C.sub.α -C.sub.α distances between residue pairs in themodeled B3 at positions homologous to those listed above are set out inTable 1, below.

Introduction of one pair of cysteine substitutions will be sufficientfor most applications. Additional substitutions may be useful anddesirable in some cases.

Modifications of the genes to encode cysteine at the target point may bereadily accomplished by well-known techniques, such as site-directedmutagenesis (see, Gillian and Smith, Gene, 8: 81-97 (1979) and Roberts,et al, Nature, 328:731-734 (1987)) by the method described in Kunkel,Proc. Natl. Acad. Sci. USA 82:488-492 (1985), or by any other meansknown in the art.

Separate vectors with sequences for the desired V_(H) and V_(L)sequences (or other homologous V sequences) may be made from themutagenized plasmid. The sequences encoding the heavy chain regions andthe light chain regions are produced and expressed in separate culturesin any manner known or described in the art, with the exception of theguidelines provided below. If another sequence, such as a sequence for atoxin, is to be incorporated into the expressed polypeptide, it can belinked to the V_(H) or the V_(L) sequence at either the N- or C-terminusor be inserted into other protein sequences in a suitable position. Forexample, for Pseudomonas exotoxin (PE) derived fusion proteins, eitherV_(H) or V_(L) should be linked to the N-terminus of the toxin or beinserted into domain III of PE, like for example TGFα in Theuer et al.,J. Urol., 149: 1626-1632 (1993), or inserted in place of domain Ib ofPE. For Diphtheria toxin-derived immunotoxins, V_(H) or V_(L) ispreferably linked to the C-terminus of the toxin.

Peptide linkers, such as those used in the expression of recombinantsingle chain antibodies, may be employed to link the two variableregions (V_(H) and V_(L), V_(and) V.sub.β) if desired and may positivelyincrease stability in some molecules. Bivalent or multivalent disulfidestabilized polypeptides of the invention can be constructed byconnecting two or more, preferably identical, V_(H) regions with apeptide linker and adding V_(L) as described in the examples, below.Connecting two or more V_(H) regions by linkers is preferred toconnecting V_(L) regions by linkers since the tendency to formhomodimers is greater with V_(L) regions. Peptide linkers and their useare well-known in the art. See, e.g., Huston et al., Proc. Natl. Acad.Sci. USA, supra; Bird et al., Science, supra; Glockshuber et al., supra;U.S. Pat. No. 4,946,778, U.S. Pat. No. 5,132,405 and most recently inStemmer et al., Biotechniques 14:256-265 (1993).

C) Various dsFv fragment molecules.

It should be understood that the description of the dsFv peptidesdescribed above can cover all classes/groups of antibodies of alldifferent species (e.g., mouse, rabbit, goat, human) chimeric peptides,humanized antibodies and the like. "Chimeric antibodies" or "chimericpeptides" refer to those antibodies or antibody peptides wherein oneportion of the peptide has an amino acid sequence that is derived from,or is homologous to, a corresponding sequence in an antibody or peptidederived from a first gene source, while the remaining segment of thechain(s) is homologous to corresponding sequences of another genesource. For example, chimeric antibodies can include antibodies wherethe framework and complementarity determining regions are from differentsources. For example, non-human CDRs are integrated into human frameworkregions linked to a human constant region to make "humanizedantibodies." See, for example, PCT Application Publication No. WO87/02671, U.S. Pat. No. 4,816,567, EP Patent Application 0173494, Jones,et al., Nature, 321:522-525 (1986) and Verhoeyen, et al., Science,239:1534-1536 (1988). Similarly, the source of V_(H) can differ from thesource of V_(L).

Particularly preferred binding agents are derived from antibodies thatspecifically recognize and bind to receptors or other surface markerscharacteristic of cancer cells. Such markers, and correspondingantibodies are well known to those of skill and include, but are notlimited to carcinoembryonic antigen (CEA), the transferrin receptor(targeted by HB21), the EGF receptor (targeted by TGFα), P-glycoprotein,c-erbB2 (targeted by e23), Lewis^(Y) carbohydrate antigens (targeted byB1, B3, B5, BR96, etc.), the IL-2 receptor (targeted by anti-Tac), andantigens described in the Abstracts of the Third InternationalConference on Monoclonal Antibody Immunoconjugates for Cancer (SanDiego, Calif. 1988).

D) Molecules homologous to antibody Fv domains--T-cell receptors.

This binding agents used in this invention can be derived from moleculesthat exhibit a high degree of homology to the antibody Fv domains,including the ligand-specific V-region of the T-cell receptor (TCR). Anexample of such an application is outlined below. The sequence of theantigen-specific V region of a TCR molecule, 2B4 (Becker et al, Nature(London) 317: 430-434 (1985)), was aligned against the Fv domains of twoantibody molecules McPC603 (see below) and J539 (Protein Data Bank entry2FBJ), using a standard sequence alignment package. When the V.sub.αsequence of 2B4 was aligned to the V_(H) sequences of the twoantibodies, the S1 site residue, corresponding to V_(H) 44 of B3, can beidentified as V.sub.α 43S (TCR 42 in the numbering scheme of Kabat andWu) and the S2 site residue, corresponding to V_(H) 111 of B3, asV.sub.α 104Q (TCR 108 in the numbering scheme of Kabat and Wu). When thesame V.sub.α sequence was aligned to the V_(L) sequences of the twoantibodies, the same residues, V.sub.α 43S and V.sub.α 104Q, can beidentified, this time aligned to the residues corresponding to V_(L) 48and V_(L) 105 of B3, respectively. Similarly, the 2B4 residues V.sub.β42E and Vβ107P (TCR 42 and 110 in the numbering scheme of Kabat, et al.)can be aligned to antibody residues corresponding to V_(H) 44 and V_(H)111 of B3 and at the same time to V_(L) 48 and V_(L) 105 of B3.Therefore, the two most preferred interchain disulfide bond sites inthis TCR are V.sub.α 43 - V.sub.β 107 and V.sub.α 104 - V.sub.β 42.Mutating the two residues in one of these pairs of residues intocysteine will introduce a disulfide bond between the α and β chains ofthis molecule. The stabilization that results from this disulfide bondwill make it possible to isolate and purify these molecules in largequantities.

II. Modified Toxins

As indicated above, the preferred immunotoxins comprise adisulfide-stabilized binding agent joined to a Pseudomonas exotoxinmodified (e.g. truncated) so that proteolytic cleavage is not requiredfor cytotoxic activity. As used herein, cytotoxic activity refers to theability to kill a cell or to significantly reduce its growth orproliferation rate.

The PE molecules of this invention are uniquely characterized by theirincreased cytotoxicity to target cells and increased antitumor activitywhen coupled with a disulfide-stabilized binding agent specific for thetarget cells. The increased cytotoxicity occurs in comparison to the useof native fusion proteins (comprising a PE that does require proteolyticcleavage) joined to a disulfide stabilized binding agent (see, e.g.commonly assigned U.S. Ser. No. 08/077,252, filed on Jun. 14, 1993) orin comparison to fusion proteins comprising a modified PE that does notrequire proteolytic activation fused to a single chain Fv (scFv) (see,e.g. commonly assigned U.S. Ser. No. 08/405,615, filed on Mar. 15,1995).

Assays for determining cytotoxicity typically involve a comparisonbetween the fusion protein comprising the subject PE molecule and adisulfide-stabilized binding agent and a fusion protein comprising areference PE molecule, e.g. PB40, joined to a disulfide-stabilizedbinding agent or conversely a modified PE molecule joined to a singlechain Fv (scFv). The respective fusion proteins are then tested incytotoxicity assays against cells specific for the binding agent. IC₅₀ s(defined below) obtained may be adjusted to obtain a cytotoxicity indexby adjusting the values such that the concentration of toxin thatdisplaces 50% of labeled ligand from ligand receptors is divided by theIC₅₀ of the recombinant toxin on cells bearing the ligand receptors. Thecytotoxicity index for each PE molecule is then compared.

PE molecules having corrected cytotoxicity indices of about 20 times ormore, preferably about 60 times or more, and most preferably about 300times or more, over PE40 or other PE molecules where no deletion ofdomain II has occurred are desired. A PE molecule lacking domain Ia maybe expressed by plasmid pJH8 which expresses domains II, Ib and III.Plasmid pJH8 is described in U.S. Pat. No. 4,892,827 and is availablefrom the American Type Culture Collection in Rockville, Md. as ATCC67208.

"IC₅₀ " refers to the concentration of the toxin that inhibits proteinsynthesis in the target cells by 50%, which is typically measured bystandard ³ H-leucine incorporation assays. Displacement assays orcompetitive binding assays are well known and described in the art. Theymeasure the ability of one peptide to compete with another peptide forthe binding of a target antigen.

A preferred PE molecule is one in which domain Ia is deleted and no morethan the first 27 amino acids have been deleted from the amino terminalend of domain II. This substantially represents the deletion of aminoacids 1 to 279. The cytotoxic advantage created by this deletion isgreatly decreased if the following deletions are made: 1-281; 1-283;1-286; and 314-380. It is surprising that the deletion of 27, but not29, 31, 33 or 36 amino acids from the amino end of domain II results inincreased toxic activity since this domain is responsible for thetranslocation of the toxin into the cytosol.

In addition, the PE molecules can be further modified usingsite-directed mutagenesis or other techniques known in the art, to alterthe molecule for particular desired application. Means to alter the PEmolecule in a manner that does not substantially affect the functionaladvantages provided by the PE molecules described here can also be usedand such resulting molecules are intended to be covered herein.

For maximum cytotoxic properties of a preferred PE molecule, severalmodifications to the molecule are recommended. An appropriate carboxylterminal sequence to the recombinant molecule is preferred totranslocate the molecule into the cytosol of target cells. Amino acidsequences which have been found to be effective include, REDX (SEQ IDNO:8) (as in native PE), REDL (SEQ ID NO:13) or KDEL (SEQ ID NO:9),repeats of those, or other sequences that function to maintain orrecycle proteins into the endoplasmic reticulum, referred to here as"endoplasmic retention sequences". See, for example, Chaudhary et al,Proc. Natl. Acad. Sci. USA 87:308-312 and Seetharam et al, J. Biol Chem.266: 17376-17381 (1991) and commonly assigned, U.S. Ser. No. 07/459,635filed January 2, 1990).

Deletions of amino acids 365-380 of domain Ib can be made without lossof activity. Further, a substitution of methionine at amino acidposition 280 in place of glycine to allow the synthesis of the proteinto begin and of serine at amino acid position 287 in place of cysteineto prevent formation of improper disulfide bonds is beneficial.

As an alternative to deletion, domain Ib can be substituted with thedisulfide stabilized binding agent as described above and in Example 1.

III. Protein Expression and Purification

The fusion proteins of this invention can be produced according to anumber of means well known to those of skill in the art. Where thedisulfide-stabilized binding agent and/or the modified Pseudomonasexotoxin are relatively short (i.e., less than about 50 amino acids)they may be synthesized using standard chemical peptide synthesistechniques. Where both molecules are relatively short the chimericmolecule may be synthesized as a single contiguous polypeptide.Alternatively the targeting molecule and the effector molecule may besynthesized separately and then fused by condensation of the aminoterminus of one molecule with the carboxyl terminus of the othermolecule thereby forming a peptide bond. Alternatively, the targetingand effector molecules may each be condensed with one end of a peptidespacer molecule thereby forming a contiguous fusion protein.

Solid phase synthesis in which the C-termninal amino acid of thesequence is attached to an insoluble support followed by sequentialaddition of the remaining amino acids in the sequence is the preferredmethod for the chemical synthesis of the polypeptides of this invention.Techniques for solid phase synthesis are described by Barany andMerrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides:Analysis, Synthesis, Biology. Vol. 2: Special Methods in PeptideSynthesis, Part A.,Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156(1963), and Stewart et al, Solid Phase Peptide Synthesis, 2nd ed. PierceChem. Co., Rockford, Ill. (1984) which are incorporated herein byreference.

In a preferred embodiment, the chimeric fusion proteins of the presentinvention are synthesized using recombinant DNA methodology. Generallythis involves creating a DNA sequence that encodes the fusion protein,placing the DNA in an expression cassette under the control of aparticular promoter, expressing the protein in a host, isolating theexpressed protein and, if required, renaturing the protein.

DNA encoding the fusion proteins (e.g. PE35/e23(dsFv)KDEL,B1(dsFv)-PE33, etc.) of this invention may be prepared by any suitablemethod, including, for example, cloning and restriction of appropriatesequences or direct chemical synthesis by methods such as thephosphotriester method of Narang et al. Meth. Enzymol 68: 90-99 (1979);the phosphodiester method of Brown et al, Meth. Enzwmol 68: 109-151(1979); the diethylphosphoramidite method of Beaucage et al., Tetra.Lett., 22: 1859-1862 (1981); and the solid support method of U.S. Pat.No. 4,458,066, all incorporated by reference herein.

Chemical synthesis produces a single stranded oligonucleotide. This maybe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Alternatively, subsequences may be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes. Thefragments may then be ligated to produce the desired DNA sequence.

In a preferred embodiment, DNA encoding fusion proteins of the presentinvention may be cloned using DNA amplification methods such aspolymerase chain reaction (PCR). Thus, for example, in a preferredembodiment, B1(V_(H))R44C DNA was PCR amplified, using primers thatcreate a peptide linker (GGGGS; SEQ ID NO:12) at the 5' end of V_(H)along with a Bam HI, and another peptide linker (e.g.KASGGPE; SEQ IDNO:11) at the 3' end along with a HindIII restriction site. Theresulting DNA was then used to replace domain Ib of PE37 (pDF₁) by sitedirected mutagenesis to make pCTK104 encoding B1(V_(H) R44C)PE33.

While the two molecules are preferably essentially directly joinedtogether, one of skill will appreciate that the molecules may beseparated by a peptide spacer consisting of one or more amino acids.Generally the spacer will have no specific ibiological activity otherthan to join the proteins or to preserve some minimum distance or otherspatial relationship between them. However, the constituent amino acidsof the spacer may be selected to influence some property of the moleculesuch as the folding, net charge, or hydrophobicity.

Proteins of the invention can be expressed in a variety of host cells,including E. coli, and other bacterial hosts. The recombinant proteingene will be operably linked to appropriate expression control sequencesfor each host. For E. coli this includes a promoter such as the T7, trp,tac, lac or lambda promoters, a ribosome binding site, and preferably atranscription termination signal. For eukaryotic cells, the controlsequences will include a promoter and preferably an enhancer derivedfrom immunoglobulin genes, SV40, cytomegalovirus, etc., and apolyadenylation sequence, and may include splice donor and acceptorsequences. The plasmids of the invention can be transferred into thechosen host cell by well-known methods such as calcium chloridetransformation for E. coli and calcium phosphate treatment orelectroporation for mammalian cells. Cells transformed by the plasmidscan be selected by resistance to antibiotics conferred by genescontained on the plasmids, such as the amp, gpt, neo and hyg genes.

Methods for expressing polypeptides and/or refolding to an appropriatefolded form, including disulfide-stabilized binding agents andimmunotoxins from bacteria such as E. coli have been described, arewell-known and are applicable to the polypeptides of this invention.See, Buchner et al., Analytical Biochemistry 205:263-270 (1992);Pluckthun, Biotechnology, 9:545 (1991); Huse, et al., Science, 246:1275(1989) and Ward, et al., Nature, 341:544 (1989)).

Often, functional protein from E. coli or other bacteria is generatedfrom inclusion bodies and requires the solubilization of the proteinusing strong denaturants, and subsequent refolding. In thesolubilization step, a reducing agent must be present to dissolvedisulfide bonds as is well-known in the art. An exemplary buffer with areducing agent is: 0.1 M Tris, pH8, 6M guanidine, 2 mM EDTA, 0.3 M DTE(dithioerythritol). Reoxidation of protein disulfide bonds can beeffectively catalyzed in the presence of low molecular weight thiolreagents in reduced and oxidized form, as described in Saxena et al.,Biochemistry 9: 5015-5021 (1970), and especially described by Buchner,et al., Anal. Biochem., supra (1992).

Renaturation is typically accomplished by dilution (e.g. 100-fold) ofthe denatured and reduced protein into refolding buffer. An exemplarybuffer is 0.1 M Tris, pH8.0, 0.5 M L-arginine, 8 mM oxidized glutathione(GSSG), and 2 mM EDTA.

In a preferred modification to the single chain antibody protocol, theheavy and light chain regions of the disulfide-stabilized binding agentwere separately solubilized and reduced and then combined in therefolding solution. A preferred yield is obtained when these twoproteins are mixed in a molar ratio such that a molar excess of oneprotein over the other does not exceed a 5 fold excess.

It is desirable to add excess oxidized glutathione or other oxidizinglow molecular weight compounds to the refolding solution after theredox-shuffling is completed. Alternatively, the final oxidation couldbe omitted and the refolding carried out at pH 9.5.

Once expressed, the recombinant proteins can be purified according tostandard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, and the like(see, generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y.(1982) and Deutscher, M. P. Methods in Enzymology Vol. 182: Guide toProtein Purification, Academic Press, Inc. N.Y. (1990)). In a preferredembodiment, folded disulfide-stabilized and immunotoxins are purified bysequential ion exchange (Q-Sepharose and Mono Q) followed by sizeexclusion chromatography on a TSK G3000SW (Toso Haas) column.Substantially pure compositions of at least about 90 to 95 % homogeneityare preferred, and 98 to 99% or more homogeneity are most preferred forpharmaceutical uses. Once purified, partially or to homogeneity asdesired, the polypeptides should be substantially free of endotoxin forpharmaceutical purposes and may then be used therapeutically.

IV. Binding Affinity of dsFv Polypeptides

The immunotoxins of this invention are capable of specifically binding atarget molecule. For this invention, a polypeptide specifically bindinga ligand generally refers to a molecule capable of reacting with orotherwise recognizing or binding a marker (e.g. antigen or receptor) ona target cell. An antibody or other polypeptide has binding affinity fora ligand or is specific for a ligand if the antibody or peptide binds oris capable of binding the ligand as measured or determined by standardantibody-antigen or ligand-receptor assays, for example, competitiveassays, saturation assays, or standard immunoassays such as EUSA or RIA.This definition of specificity applies to single heavy and/or lightchains, CDRs, fusion proteins or fragments of heavy and/or light chains,that are specific for the ligand if they bind the ligand alone or incombination.

In competition assays the ability of an antibody or peptide fragment tobind a target molecule is determined by detecting the ability of thepeptide to compete with the binding of a compound known to the targetmolecule. Numerous types of competitive assays are known and arediscussed herein. Alternatively, assays that measure binding of a testcompound in the absence of an inhibitor may also be used. For instance,the ability of a molecule or other compound to bind the target moleculecan be detected by labelling the molecule of interest directly or themolecule be unlabelled and detected indirectly using various sandwichassay formats. Numerous types of binding assays such as competitivebinding assays are known (see, e.g., U.S. Pat. Nos. 3,376,110,4,016,043, and Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Publications, N.Y. (1988)). Assays for measuring bindingof a test compound to one component alone rather than using acompetition assay are also available. For instance, immunoglobulinpolypeptides can be used to identify the presence of the binding ligand.Standard procedures for monoclonal antibody assays, such as ELISA, maybe used (see, Harlow and Lane, supra). For a review of various signalproducing systems which may be used, see, U.S. Pat. No. 4,391,904.

V. Pharmaceutical Compositions

The recombinant fusion proteins and pharmaceutical compositions of thisinvention are particularly useful for parenteral administration, such asintravenous administration or administration into a body cavity or lumenof an organ. The compositions for administration will commonly comprisea solution of the PE molecule fusion protein dissolved in apharmaceutically acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers can be used, e.g., buffered saline and thelike. These solutions are sterile and generally free of undesirablematter. These compositions may be sterilized by conventional, well knownsterilization techniques. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example, sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate and the like. Theconcentration of fusion protein in these formulations can vary widely,and will be selected primarily based on fluid volumes, viscosities, bodyweight and the like in accordance with the particular mode ofadministration selected and the patient's needs.

Thus, a typical pharmaceutical composition for intravenousadministration would be about 0.1 to 10 mg per patient per day. Dosagesfrom 0.1 up to about 100 mg per patient per day may be used,particularly when the drug is administered to a secluded site and notinto the blood stream, such as into a body cavity or into a lumen of anorgan. Actual methods for preparing parenterally administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington'sPharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa.(1980).

The compositions containing the present fusion proteins or a cocktailthereof (i.e., with other proteins) can be administered for therapeutictreatments. In therapeutic applications, compositions are administeredto a patient suffering from a disease, in an amount sufficient to cureor at least partially arrest the disease and its complications. Anamount adequate to accomplish this is defined as a "therapeuticallyeffective dose." Amounts effective for this use will depend upon theseverity of the disease and the general state of the patient's health.

Single or multiple administrations of the compositions may beadministered depending on the dosage and frequency as required andtolerated by the patient. In any event, the composition should provide asufficient quantity of the proteins of this invention to effectivelytreat the patient.

Among various uses of the recombinant fusion proteins of the presentinvention are included a variety of disease conditions caused byspecific human cells that may be eliminated by the toxic action of theprotein. One preferred application is the treatment of cancer, such asby the use of immunotoxins comprising disulfide-stabilized bindingagents that specifically target and bind tumor markers. Such bindingagents include antibodies that bind antigens (markers) found on cancercells. Such targets are well known to those of skill in the art andinclude, but are not limited to carcinoembryonic antigen (CEA), thetransferrin receptor (targeted by TGFα), P-glycoprotein, c-erbB2(targeted by e23), Lewis^(Y) carbohydrate antigens (targeted by B1, B3,B5, BR96, etc.) and antigens described in the Abstracts of the ThirdInternational Conference on Monoclonal Antibody Immunoconjugates forCancer (San Diego, Calif. 1988).

Other applications include the treatment of autoimmune conditions suchas graft-versus-host disease, organ transplant rejection, type Idiabetes, multiple sclerosis, rheumatoid arthritis, systemic lupuserythematosus, myasthenia gravis and the like caused by T and B cells.The fusion proteins may also be used in vitro, for example, in theelimination of harmful cells from bone marrow before transplant. Theligand binding agent portion of the fusion protein is chosen accordingto the intended use. Proteins on the membranes of T cells that may serveas targets for the binding agent include CD2 (T11), CD3, CD4 and CD8.Proteins found predominantly on B cells that might serve as targetsinclude CD10 (CALLA antigen), CD19 and CD20. CD45 is a possible targetthat occurs broadly on lymphoid cells. These and other possible targetlymphocyte antigens for the binding agent are described in LeucocyteTyping III, A. J. McMichael, ed., Oxford University Press, 1987.

Those skilled in the art will realize that ligand binding agents may bechosen that bind to receptors expressed on still other types of cells asdescribed above, for example, membrane glycoproteins or growth factor orhormone receptors such as epidermal growth factor receptor and the like.

VI. Transport of Antibodies into the Cytosol

In another embodiment, this invention provides compositions and methodsfor transporting antibodies into the cytosol of the cell. The antibodiesthus transported may be selected to bind to particular intracellularcomponents (e.g. particular proteins in signal transduction systems,cytoskeletal elements, particular target RNAs, and the like). The boundantibodies can inhibit the normal activity of the target molecule andcan thus be used to selectively "knock out" particular intracellularfunctions. Depending on the antibody target this may prove cytotoxic, ormay simply alter the activity of the cell.

Thus, for example, in one embodiment, the antibody V_(H) or V_(L) mayspecifically bind and inhibit an RNA transcription product of anoncogene, thus preventing transformation of the target cell.Alternatively, the antibody may simply act as a label for detection ofthe particular intracellular component to which it binds.

Compositions for the intracellular delivery of the antibody arepreferably fusion proteins formed by joining a Pseudomonas exotoxin toan antibody fragment, more preferably to a V_(H) or a V_(L) antibodyfragment. The Pseudomonas exotoxin is preferably truncated, but stillincludes a functional translocation domain (domain II).

In a preferred embodiment, the antibody is located in domain II or IIIof the PE. Domain III, having the ADP ribosylation activity must beinactivated (e.g., by truncation, insertion of a foreign peptidesequence, or through complete elimination of domain) so that onlyantibody binding effects are manifested.

In a particularly preferred embodiment, the antibody variable domain,either heavy or light chain, should be located in domain II or III of atruncated PE which does not require proteolytic activation. Thus, forexample, in B1(V_(H))PE33, or PE35/e23(V_(H))KDEL, the V_(H) insert isnot removed by proteolysis, but is translocated along with domain II andIII of PE.

EXAMPLES

The following examples are offered to illustrate, but not to limit thepresent invention.

Example 1 Preparation and Testing of B1(dsFv)-PE33

Monoclonal antibody (MAb) B1 is a murine antibody directed againstLewis^(Y) (Le^(Y)) and related carbohydrate antigens which are abundanton the surface of many carcinomas (Pastan et al., Cancer Res. 51,3781-3787 (1991)). MAb B1 has been used to make both single-chain anddisulfide-stabilized Fv immunotoxins (Pastan et al., Cancer Res. 51,3781-3787 (1991), Benhar, et al., Prot. Eng., 7: 1509-1515 (1995), andBenhar et al. Clin. Cancer. Res., 1: 1023-1029 (1995)). These agents arecapable of causing complete regressions of established xenografts innude mice.

To achieve the goal of developing a recombinant immunotoxin that issmall, stable and does not need proteolytic processing, domain Ib (aminoacids 365-394) of PE37 (a truncated form of PE [residues 280 through613] that only contains the portion of the toxin that undergoestranslocation to the cytosol) was replaced with the V_(H) fragment ofMAb B1 linked to the V_(L) domain with a disulfide bond (FIG. 1). Asillustrated herein, the resulting molecule, B1(dsFv)-PE33 is more activethan any previous MAb B1 containing immunotoxins.

A) Construction of Plasmids for Expression of B1(dsFv)-PE33

In order to construct an active recombinant immunotoxin that was smallerthan the current generation of recombinant immunotoxins and that did notneed intracellular proteolytic cleavage for activation, the antibodyfragment B1(dsFv) was inserted between domains II and III of aPseudomonas exotoxin. This was accomplished by substituting B1(dsFv) fordomain Ib of PE37, a truncated form of PE that contains only the portionof the toxin that undergoes translocation to the cytosol. In particular,B1(V_(H))R44C was inserted after amino acid 364 of PE and the insert waspreceded by a small flexible peptide linker GGGGS (SEQ ID NO:12).Following the V_(H) domain was another peptide, KASGGPE (SEQ ID NO:11)(C3 connector) (Brinkmann et al., Proc. Natl. Acad. Sci. USA, 89:3075-3079 (1992)), connecting the carboxyl terminus of V_(H) to aminoacid 395 of the Pseudomonas exotoxin.

As shown in FIG. 1, the V_(H) domain replaced amino acids 365 to 394 ofPE37 and the V_(L) domain was connected to the V_(H) domain by adisulfide bond engineered into the framework region, with cysteinesintroduced at position 44 of the V_(H) and position 105 of V_(L)(Brinkmann et al., Proc. Natl. Acad. Sci. USA, 90: 7538-7542 (1993)).The resulting recombinant immunotoxin, termed B1(dsFv)-PE33, is 5 kDasmaller than B1(dsFv)PE38 or B1(Fv)-PE38 (FIG. 1). In the toxin portion,cysteine 287 was changed to a serine to reduce the chance of incorrectdisulfide bond formation (Theuer et al., J. Urol. 149: 1626-1632(1993)).

The construction of plasmids pDF1, which encodes PE37, which starts atmethionine followed by PE amino acids 281-613 (a truncated form of PEthat does not require proteolytic activation), and pB1V_(H) R44C-PE38which encodes the single-domain B1(V_(H))R44C-PE38 immunotoxin have beendescribed (Theuer et al., J. Biol Chem. 267: 16872-16877 (1992), Benharet al. Clin. Cancer Res. 1: 1023-1029 (1995)). Sticky feet-directedmutagenesis (Clackson et al., Nucl. Acids Res. 17: 10163-10170 (1989))using uracil-containing pDF1 as a template was used to construct theexpression plasmid encoding for B1(V_(H))R44C-PE33, the component of theintramolecularly-inserted dsFv-immunotoxin. The B1(V_(H))R44C DNA wasPCR amplified using the plasmid pB1V_(H) R44C-PE38 as a template andoligo primers CT119 with 5'-phosphorylated CT120. The forward PCR primerCT119: 5'-GGCAACGACGAGGCCGGCGCGGCCAACGGCGGTGGCGGATCCGAGGTGCAGCTGGTGGAATCTGGA3' (SEQ. ID NO:41)had sequences that are identical to the DNA encoding for PE residues356-364 and a peptide linker GGGGS inserted at the 5' end of V_(H) and aBamHI site was created (underlined). The reverse PCR oligonucleotideprimer CT120: 5'-GTCGCCGA GGAACTCCGCGCCAGTGGGCTCGGGACCTCCGGAAGCT TITGC-3' (SEQ ID NO:2) and sequences that are complementary to the DNAencoding for PE residues 395-403 and a Fv-toxin junction (connector)inserted at the 3' end of V_(H) and a HindIII site was created(underlined).

The PCR product was purified and annealed to a uracil-containingsingle-stranded DNA prepared by the rescue of PDF1 phagemid with anM13K07 helper phage (Bio-Rad). The DNA was extended and ligatedaccording to the MUTA-GENE mutagenesis kit (Bio-Rad). Because theannealing efficiency of the PCR fragment to the single-stranded templateand the mutagenesis efficiency were low (˜10%), the DNA pool used in themutagenesis reaction was digested with a restriction endonuclease whichcuts an unique site in domain Ib region but not in B1(V_(H)). This extradigestion step increased the mutagenesis efficiency to more than 50%.

Correct clones were identified by DNA restriction analysis and verifiedby DNA sequencing. The resulting immunotoxin clone was namedpB1(V_(H))R44C-PE33 or pCTK104, which encodes a single-domain B1(V_(H))immunotoxin in which the V_(H) domain is replaced for the domain Ibregion (amino acids 365 to 394) of PE37. The plasmid pB1V_(L) A105CSTOPencodes B1(V_(L))A105C, which is a component of dsFv-immunotoxin asdescribed previously (Benhar, et al. Clin. Cancer Res., 1: 1023-1029(1995)).

B) Production of Recombinant Immunotoxin

The components of the disulfide-stabilized immunotoxinsB1(V_(H))R44C-PE38,B1(V_(H))R44C-PE33, B1(V_(L))A105C, or single-chainimmunotoxin B1(Fv)-PE38 were expressed in separate E. coli BL21(λDE3)(Studier,et al., J. Mol Biol., 189: 113-130 (1986)) cultures harboringthe corresponding expression plasmid. All recombinant proteinsaccumulated in inclusion bodies. Disulfide stabilized immunotoxins wereobtained by mixing equimolar amounts of solubilized and reducedinclusion bodies essentially as described (Reiter et al., Cancer Res.,54: 2714-2718 (1994)), except that the final oxidation step was omittedand refolding was carried out at pH 9.5. Properly foldeddisulfide-stabilized and single-chain immunotoxins were purified bysequential ion exchange (Q-Sepharose and Mono Q) followed by sizeexclusion chromatography on a TSK G3000SW (Toso Haas) column.

The proteins obtained were over 95 % homogeneous and had the expectedmolecular mass of 59 kDa on SDS-PAGE. In the presence of the reducingagent β-mercaptoethanol, the dsFv-immunotoxin, B1(dsFv)-PE33, wasreduced into two species; one is B1(VL105C) and the other is B1(V)-PE33.The apparent molecular weights of these components are 13 kDa and 46kDa, respectively. The single-domain B1(V_(H))-PE33 immunotoxin was alsomade and purified. The yield of either B1(dsFv)-PE33 or B1(V_(H))-PE33was 8-10% of the protein present in inclusion bodies.

C) Cytotoxic Activity of B1(dsFv)-PE33 Toward B1-antigen Expressing CellLines

The cytotoxicity of B1(dsFv)-PE33 was determined by measuring thereduction in the incorporation of (³ H)-leucine by various human cancercell lines after treatment with immunotoxin (Kuan et al. J. Biol. Chem.,269: 7 B1(dsFv)-PE38 and B1(V,)-PE33 (no light chain) were included forcomparison. Table 1 shows that all three proteins are cytotoxic to cellsexpressing B1antigen (e.g. A431, MCF7, CRL1739, and LNCaP) but not tocells that do not bind MAb B1 (e.g. L929 and HUT102).

                                      TABLE 1                                     __________________________________________________________________________    Cytotoxicity of B1 immunotoxins toward various cell lines.                                Antigen.sup.3                                                                       Cytotoxicity.sup.1 IC.sub.50 ng/ml                          Cell Line.sup.2                                                                    Cancer type                                                                          Expression                                                                          B1(dsFv)PE38                                                                          B1(dsFv)P33                                                                         B1(V.sub.H)PE33                               __________________________________________________________________________    A431 epidermoid                                                                           +++   0.5     0.25  2.0                                           MCF7 breast +++   0.9     0.35  4.0                                                carcinoma                                                                CRL1739                                                                            gastric                                                                              +++   0.4     0.31  N.D..sup.4                                    LNCaP                                                                              prostate                                                                             +     7.0     1.3   N.D..sup.4                                    HUT102                                                                             T-cell -     >1000   >1000 >1000                                              leukemia                                                                 L929 mouse  -     >1000   >1000 >1000                                              fibroblast                                                               __________________________________________________________________________     .sup.1 Cytotoxcity data are given as IC.sub.50 values, where IC.sub.50 is     the concentration of immunotoxin that causes a 50% inhibition of protein      synthesis after a 20 hour incubation with the immunotoxin.                    .sup.2 All of the cell lines except L929 are of human origin.                 .sup.3 The level of antigen is marked +++, + and - for strong, medium and     no detectable expression respectively.                                        .sup.4 Not determined.                                                   

In this assay, B1(dsFv)-PE33 had an IC₅₀ of 0.25 ng/ml on A431 cells and0.35 ng/ml on MCF7 cells. B1(dsFv)-PE33 was more active to allantigen-positive cell lines in this study than B1(dsFv)-PE38 whichrequires processing proteolysis. To analyze whether the cytotoxicity ofB1(dsFv)-PE33 was specific, competition experiments were carried outwith an excess of MAb B1.

The resulting data showed that the intoxication of A431 carcinoma cellsby B1(dsFv)-PE33 was due to the specific binding to the B1 antigen,since its cytotoxicity was blocked by excess MAb B1. B1(V_(H))-PE33 thatwas not associated with light chain was also tested and it proved to beabout 10-fold less cytotoxic (IC₅₀ 2 ng/ml on A431 cells) thanB1(dsFv)-PE33 (Table 1) indicating the heavy chain has a large role inantigen binding. However, a related single-domain immunotoxin(B3(V_(H))-PE38) which requires proteolytic processing for activationwas much less active with an IC₅₀ of 40 ng/ml on A431 cells (Brinkmaannet al., J. Immunol. 150, 2774-2782 (1993)).

D) Antigen Binding of B1(dsFv)-PE33

To determine whether the improved cytotoxicity of B1(dsFv)-PE33 was dueto improved binding or some other factor, the antigen binding affinityof B1(dsFv)-PE33 on antigen-positive cells (e.g., A431 cells) determinedby competition assays, in which increasing concentrations of eachimmunotoxin competed for the binding of (¹²⁵ I)-B1-IgG to A431 cells at4° C. B1IgG, B1(dsFv)-PE38, B1(dsFv)-PE33 and B1(V_(H))-PE33 competedfor the binding of (₁₂₄ I)-B1-IgG to A431 cells by 50% at 40 nM, 2 mM,3.5 mM, and 25 mM, respectively. Thus, the binding affinity ofB1(dsFv)-PE33 was slightly less than B1(dsFv)-PE38 suggesting that theimproved cytotoxicity was not due to improved binding, but rather thatelimination of the requirement for proteolytic activation wasresponsible for the improved cytotoxicity. The single-domain immunotoxinB1(V_(H))- P33 exhibited a 10-fold lower binding affinity relative tothe dsFv-immunotoxins consistent with its diminished cytotoxicity (Table1).

E) Stability of B1(dsFv)-PE33

Thermal stability of the immunotoxins was determined by incubating themat 100 μg/ml in PBS at 37° C. for 2 or 8 hours, followed by analyticalchromatography on a TSK G3000SW (Toso Haas) column to separate themonomers from larger aggregates (Reiter et al. Protein Eng., 7: 697-704(1994)). Relative binding affinities of the immunotoxins were determinedby adding ¹²⁵ I-labeled B1-IgG to 10 A431 cells as a tracer with variousconcentrations of the competitor. The binding assays were performed at4° C. for 2 h in RPMI containing 1% bovine serum albumin and 50 mM MES(Sigma) as described (Batra et al., Proc. Natl. Acad. Sci. USA 89:5867-5871 (1992)).

Both B1(dsFv)-PE33 and B1(dsFv)-PE38 were monomers before incubation inPBS at 37° C. and remained monomeric for 2 or 8 hrs. In contrast, thesingle-chain immunotoxin B1(Fv)PE38 formed >60% aggregates after an 8 hrincubation at 37° C. (Table 2, see also et al., Clin. Cancer Res., 1:1023-1029 (1995)). Following the 8 hr incubation at 37° C.,B1(dsFv)-PE33 and B1(dsFv)-PE38 retained almost all its initialcytotoxic activity as before incubation, while B1(Fv)-PE38 lost 75% ofits cytotoxic activity. Thus, both B1(dsFv)-PE38 and B1(dsFv)-PE33 areextremely stable at 37° C. presumably because they do not tend todenature and aggregate as do the scFV immunotoxins.

F) Toxicity and Antitumor Activity in Nude Mice

The single dose mouse LD₅₀ was determined using female BALB-c mice (6-8weeks old˜20 gm) which were given a single i.v. injection of differentdoses of B1(dsFv)PE38 or B1(dsFv)PE33 diluted in 200 μl of PBS-HSA. Micewere followed for two weeks after injection. Athymic (Nu-Nu) mice,female 6-8 weeks old˜20 gm, were injected subcutaneously on day 0 with3×10⁶ A431 cells suspended in RPMI medium without FBS. By day 5, tumorswere about 50 to 70 mm³ in size. Mice were treated on days 5, 7, and 9by i.v. injections of different doses of immunotoxins diluted inPBS-HSA. Tumors were measured with a caliper and the tumor volumes werecalculated using the formula: volume=(length)×(width)² ×(0.4).

The LD₅₀ of both immunotoxins was found to be 0.5 mg/kg. The toxicity isthe same as the LD₅₀ value determined for the B1(Fv)-PE38 as well asother anti-Le^(y) Fv-immunotoxins (Reiter et al. Cancer Res., 54:2714-27 (1994)). The results show that even though the immunotoxin ismore active to target cells because it does not require proteolyticactivation, it is not more toxic to mice. This toxicity in mice ispresumed to be due to non-specific uptake by the liver (Keritman et al.,Blood, 83: 426-434 (1994).

G) Improved Antitumor Activity of B1(dsFv)-PE33

To determine whether the improved cytotoxicity in vitro is accompaniedby an increase in antitumor activity, B1(dsFv)-PE33 and B1(dsFv)-PE38were compared by assessing their ability to cause regressions ofestablished human carcinoma xenografts in nude mice. Nude mice received3×10⁶ A431 cells subcutaneously on day 0. Five days later, when tumorsaveraged 50-70 mm³ in volume, the mice were treated with three i.v.injections on days 5, 7, and 9 of various doses of immunotoxin. Controlmice were treated with PBS-HSA only.

As shown in FIG. 4, both immunotoxins demonstrated significantdose-dependent anti-tumor activity. B1(dsFv)-PE38 caused only partialregression of A431 tumors at the 6.5 ,μg/kg (100 pmole/kg) dose level,whereas B1(dsFv)-PE33 at the same 100 pmole/kg (6 μg/kg) dose causedcomplete disappearance of the tumors (FIG. 4). Furthermore, the tumorstreated with 200 pmole/kg (13 μg/kg) B1(dsFv)-PE38 regressed completelyafter the third injection but regrew within a few days whereas 200pmole/kg B1(dsFv)-PE33 caused complete regressions that lasted over onemonth in 5 out of 5 animals. These results indicate that B1(dsFv)-PE33has significantly better antitumor activity than B1(dsFv)-PE38. Hence,the improved cytotoxicity in vitro correlates with the improvedantitumor activity in animals.

Since both B1(dsFv)-PE33 and B1(dsFv)-PE38 have the same toxicity inmice, the PE33 version has a larger therapeutic window. The effectivedose causing complete remissions in nude mice is 2.5% of the mouse LD₅₀.This makes B1(dsFv)-PE33 a good candidate for clinical development as ananti-cancer agent. The improved antitumor activity of B1(dsFv)-PE33 overB1(dsFv)-PE38 is a consequence of better cytotoxicity in vitro, due tolack of a requirement for proteolytic activation and smaller size forbetter tumor penetration. Since the efficiency of proteolytic activationcan vary in different types of cells, this new type of recombinantimmunotoxin will prove more useful than the previous generation ofmolecules which require proteolytic activation.

In the foregoing experiments, the B1 dsFv fragment was inserted betweenthe translocation domain and ADP-ribosylation domain of PE, replacingdomain Ib. In fact, it is also possible to delete a portion of domain II(amino acids 343-364) without loss of activity. In addition, analyses ofthe proposed structure of B1(dsFv)PE33 using computer graphics showsthat the domain Ib region is a good location for insertion of dsFvfragment because the CDRs should still be free to interact with antigen.The results in the foregoing experiments indicate that the presence ofB1(dsFv) in this region -only minimally affected antigen binding to A431cells.

Example 2

Preparation and Testing of PE35/e23(dsFv)KDEL

In order to construct an active recombinant immunotoxin that was smallerthan the current generation of recombinant immunotoxins and that did notneed intracellular proteolytic cleavage for activation the e23(dsFv)antibody fragment was inserted near the carboxyl terminus of PE35KDEL, atruncated form of PE that contains only the portion of the toxin thatundergoes translocation to the cytosol (FIG. 2).

A) Construction of Plasmids

All plasmids listed in FIG. 3 use anisopropyl-l-thio-β-D-galactopyranoside-inducible 17 promoter expressionsystem (Studier & Moffatt J. Mol Biol. 189, 113-130 (1986)). PlasmidpCT12 encodes for a protein, termed PE35/TGFαKDBL, starting with a Metat position 280 of PE and amino acids 281 to 364 and 381 to 607 with agene encoding TGFα inserted between amino acids 607 and 604 of PE, andthe amino acids KDEL are substituted for the carboxyl-terminal REDLKsequence of PE (Theuer et al., J. Urol., 149: 1626-1632 (1993)). PlasmidpYR39, encoding e23(V_(H) Cys₄₄)-PE38KDEL, is the expression plasmid forthe V_(H) -Toxin components of the dsFv-immunotoxin e23(dsFv)-PE38KDEL(Reiter et al., J. Biol. Chem., 269: 18327-18331 (1994)). PlasmidspCTK101 and pCTK103 encoding PE35/e23(V_(H) Cys₄₄)KDEL andPE35/e23(V_(H) Cys₄₄) are the expression plasmids for the Toxin-V_(H)components of the dsFv-immunotoxin PE/e23(dsFv)KDEL. They wereconstructed by cloning the StuI-EcoRI digested PCR fragments intoStuI-EcoRI restriction sites in pCT12. The PCR reactions were carriedout using 10 ng of pYR39 as template and 100 pmoles of primers5'-AAACCGAGGCCTTCCGGAGGTGGTGG ATCCGAAGTGCAGCTGCAGGAGTCAGGA-3' (SEQ IDNO:3) and 5' -TTAGCAGCCGAATTCTrAGAGCTCGTCTTTCGGCGGTTGCCGGAGGAGACGGTGACCGT GGTCCCTG-3' (SeqID NO:-4) for PE35/e23(V_(H) Cys₄₄)KDEL or 5'-AAACCGAGGCCTTCCGGAGGTGGTGGATCCGAAGTGCAGCTGCAGGAGTCAGGA-3' (SEQ. ID NO: 5) and5'-GATCGCTCGGAATTCTTAGGAGACGGTGACCGTGGTC CCTGC-3' (SEQ ID NO:6) forPE35/e23(V_(H) Cys₄₄). The protein encoded by pCTK101 is a single-domainimmunotoxin in which e23(V_(H) Cys₄₄) was introduced between residue 607of PE followed by a peptide linker SGGGGS and residue 604 to 608 andKDEL. The protein encoded by pCTK103 was the same as pCTK101 encodedprotein except without amino acid 604 to 608 and KDEL.

Plasmid pYR40 encodes e23(V_(L) Cys₉₉), the V_(L) component of thedsFv-immunotoxin (Reiter et al., J. Biol. Chem. 269, 18327-18331(1994)), while pCTK102 encodes e23(V_(L) Cys₉₉) fused to PE amino acids604-608 and carboxyl terminal sequences KDEL. This plasmid wasconstructed by subcloning a NdeI-EcoRI digested PCR product, which usedpYR40 as template and T7 promoter primer as well as5'-TTAGCAGCCGAATTCTTAGAGCTCGTCTITTCGGCGGTTTGCCGGAGGAGACGGTGACCGTGGTCCCTG-3' (SEQ ID NO:7) as primers, into NdeI-EcoRIrestriction sites found in pYR40. Positions of cysteine replacement inframework region of e23(Fv) are Asn⁴⁴ ->Cys in V_(H) and Gly⁹⁹ ->Cys inV_(L) were described previously (Reiter et al., J. Biol. Chem. 269:18327-18331 (1994)). All plasmids were confirmed by DNA sequencing.

The V_(H) rather than the V_(L) was inserted near the carboxyl terminusof PE35KDEL, since PE35/e23(V_(H))KDEL (unattached to V_(L)) is lesssoluble and more likely to precipitate than PE35/e23(V_(l))KDEL notattached to V_(H) (Brinlrann et al., J. Immunol., 150: 2774-2782 (1993);Reiter et al., Biochem., 33: 5451-5459 (1994)). The disulfide bond formsbetween cysteines introduced at position 44 of the V_(H) and position 99of V_(L) (Reiter et al., J. Biol. Chem., 269: 18327-18331 (1994)). Inthe toxin portion, cysteine 287 was changed to a serine to reduce thechance of incorrect disulfide bond formation (Theuer et al., J. Urol.,149: 1626-1632 (1993); FIG. 2). The location chosen for e23 (V_(H)CYS₄₄) insertion was after amino acid 607 of PE and it was preceded by asmall peptide linker SGGGGS (SEQ ID NO:10). Following the V_(H) domainare amino acids 604-608 and KDEL (SEQ ID NO:9)(FIG. 1). A diagram ofthis molecule, PE35/e23(dsFv)KDEL (I) is shown in FIGS. 2 and 3.

B) Production of Recombinant Proteins

The components of the disulfide-stabilized immunotoxins PE35/e23(V_(H)Cys₄₄)KDEL, PE35/e23(H Cys₄₄), e23(V_(H) Cys₄₄)-PE38KDEL, e23(V_(L)Cys₉₉), and e23(V_(L) Cys₉₉)KDEL or single-chain immunotoxins wereproduced in separate E. coli BL21(1DE3) (Studier & Moffatt, J. Mol.Biol., 189: 113-130 (1986)) cultures harboring the correspondingexpression plasmid (See FIG. 3). All recombinant proteins accumulated ininclusion bodies. Properly folded disulfide stabilized immunotoxins wereobtained by mixing equimolar amounts of solubilized and reducedinclusion bodies essentially as described (Reiter et al., Cancer Res.,54: 2714-2718 (1994)), except that the final oxidation step was omittedand refolding was carried out at pH 9.5.

As shown in FIG. 3, PE35/e23(dsFv)KDEL (I) was produced by mixingPE35-e23(V_(H) Cys₄₄)KDEL and e23(V_(L) Cys₉₉); PE35/e23(dsFv)KDEL (II)was produced by mixing PE35-e23(V_(H) Cys₄₄) and e23(V_(L) Cys₉₉)KDEL;PE35/e23(dsFv)KDEL (III) was produced by mixing PE35-e23(V_(H)Cys₄₄)KDEL and e23(V_(L) Cys₉₉)KDEL; PE35/e23(dsFv) (I) was produced bymixing PE35-e23(V_(H) Cys₄₄) and e23(V_(L) Cys99). The immunotoxins werepurified by refolding of inclusion bodies in a redox-shuffling buffer.Properly folded disulfide-stabilized and single-chain immunotoxins werepurified by sequential ion exchange (Q-sepharose and Mono Q) followed bysize exclusion chromatography on a TSK G3000SW (Toso Haas) column.

The proteins obtained were over 95 % homogeneous and had the expectedmolecular mass on SDS-PAGE (60 kDa). In the presence of the reducingagent b-mercaptoethanol, the dsFv-immunotoxin, PE35/e23(dsFv)KDEL (I)was reduced into two species; one was e23(V_(L) Cys₉₉) and the other wasa single-domain toxin PE35/e23(V_(H) Cys₄₄)KDEL. The apparent molecularweights of these components was, as expected, 13 kDa and 47 kDa,respectively.

C) Specific Cytotoxic Activity of PE35/e23(dsFv)KDEL Toward e23-antigenExpressing Cell Lines

The cytotoxicity of PE35/e23(dsFv)KDEL was determined by measuring thereduction in the incorporation of [³ H]-leucine by various human cancercell lines after treatment with serial dilutions of the immunotoxin inPBS containing 0.2% HSA as described previously (Kuan et al., J. BiolChem., 269: 7610-7616 (1994)). e23(scFv)-PE38KDEL and e23(dsFv)-PE38KDELwere included for comparison. Table 2 shows that a comparison of theactivity of the immunotoxin PE35-e23(dsFv)KDEL (1) and the other tworeference molecules, e23(scFv)-PE38KDEL and e23(dsFv)-PE38KDEL,indicates that all three proteins are cytotoxic to cells expressing

                                      TABLE 2                                     __________________________________________________________________________    Cytotoxicity of e23 immunotoxins towards various cell lines.                                    Cytotoxicity.sup.1 IC.sub.50 ng/ml                                     Antigen.sup.2                                                                        e23(scFv)                                                                           e23(dsFv)                                                                            PE35/e23                                       Cell Line                                                                          Cancer type                                                                         Expression                                                                           PE38KDEL                                                                            PE38KDEL                                                                             (dsFv)KDEL (I)                                 __________________________________________________________________________    N-87 gastric                                                                             +++    0.5   0.1    0.8                                            A431 epidermoid                                                                          +      2.9   1.0    3.0                                            Hut102W                                                                            leukemia                                                                            -      >1000 >1000  >1000                                          __________________________________________________________________________     .sup.1 Cytotoxcity data are given as IC.sub.50 values, where IC.sub.50 is     the concentration of immunotoxin that causes a 50% inhibition of protein      synthesis after a 20 hour incubation with the immunotoxin.                    .sup.2 The level of antigen is marked +++, + and - for strong, medium and     no detectable expression respectively.                                   

erbB2 (e.g. N87 and A431) but not to cells (e.g. HUT-102) that do notbind MAb e23 (Table 2). In this assay, PE35/e23(dsFv)KDEL() had an IC₅₀of 0.8 ng/ml on N87 cells. Although its activity is less than the twoother molecules (IC5₀ of 0.5 ng/ml for e23(scFv)-PE38KDEL and 0.1 ng/mlfor e23(dsFv)-PE38KDEL), it is still extremely active.

D) Improved Stability of Immunotoxin PE35/e23(dsFV)KDEL (I)

Thermal stability of the immunotoxins was determined by incubating themat 100 μg/ml in PBS at 37° C. for 2 or 8 hours, followed by analyticalchromatography on a TSK G3000SW (Toso Haas) column to separate themonomers from dimers and larger aggregates. PE35/e23(dsFv)KDEL (1) was amonomer before incubation in PBS at 37° C. and remained monomeric for 2or 8 hrs. In contrast, the single-chain immunotoxin e23(Fv)PE38KDELformed 30% aggregates and 25% dimers after an 8 h incubation at 37° C.Following the 8 h 37° C. treatment, PE35/e23(dsFv)KDEL (I) retainedalmost the same cytotoxic activity as before treatment, whilee23(Fv)PE38KDEL had an IC₅₀ of 3.1 ng/ml on N-87 cells, which is only16% of its cytotoxic activity before treatment. This result indicatesthat the purified PE35/e23(dsFv)KDEL like e23(dsFv)-PE38KDEL (Reiter etal., Protein Eng., 7: 697-704 (1994)) is very stable and has a lowpropensity to aggregate.

E) Antigen-binding Analysis of PE35/e23(dsFv)KDEL (I)

To investigate the reason for the decreased cytotoxicity ofPE35/e23(dsFv)KDEL (I), its antigen binding affinity on antigen-positivecells (e.g., N87 cells) was analyzed by competition assays in whichincreasing concentrations of each immunotoxin were present to competefor the binding of [¹²⁵ I]-e23-IgG to N87 cells at 4° C. The e23 IgG,e23(dsFv)-PE38KDEL, and PE35/e23(dsFv)KDEL competed for the binding of[¹²⁵ I]-e23 IgG to N87 cell by 50% at 4 nM, 140 nM and 500 nM,respectively. Thus, the binding affinity of PE35/e23(dsFv)KDEL (1) is4-fold less than e23(dsFv)-PE38KDEL on N87 cells. Hence, the lowercytotoxicity of PE35/e23(dsFV)KDEL (1) is associated with a lowerbinding affinity. As previous reported the bivalent e23IgG had a higherapparent affinity than e23(dsFv)PE38KDEL which is monovalent (Reiter etal., J. Biol Chem., 269: 18327-18331 (1994)).

F) Importance of the Position of KDEL (SEQ ID NO:9) for Cytotoxicity

In PE35/e23(dsFv)KDEL (SEQ ID NO:9) (I), the KDEL is on the samepolypeptide chain as the toxin moiety. The KDEL sequence is consideredto mediate transport of the toxin moiety of the immunotoxin to the ERwhere it can translocate. To address whether it was important to havethe KDEL (SEQ ID NO:9) sequence on the C-terminus of the toxin, orwhether it could be attached to the C-terminus of the V_(L) which isattached to V_(H) PE35 by a disulfide bond molecules were constructedhaving KDEL (SEQ ID NO:9) on V_(L) instead of the V_(H) toxin, with KDEL(SEQ ID NO:9) on both the V_(H) -toxin and the V_(L) and with KDEL (SEQID NO:9) on neither (FIG. 2). These were termed PE35/e23(dsFv)KDEL II-IV(Table 3 and FIG. 2). Table 2 shows that for the recombinant toxin toinhibit protein synthesis on target cells, it is

                  TABLE 3                                                         ______________________________________                                        Comparison of four different types of PE35/e23(dsFv)KDEL.                                     Activity.sup.1                                                                          Relative binding.sup.1,2                            Construct       IC.sub.50 (ng/ml)                                                                       (nM)                                                ______________________________________                                        PE35/e23(dsFv)KDEL(I)                                                                         0.8       500                                                 PE35/e23(dsFv)KDEL(II)                                                                        1000      400                                                 PE35/e23(dsFv)KDFL(IV)                                                                        1.2       530                                                 PE35/e23(dsFv)(IV)                                                                            >1000     610                                                 ______________________________________                                         .sup.1 Cytotoxicity and binding assays were measured on N87 cell line.        .sup.2 The concentration of competitor which caused 50% inhibition of the     binding of .sup.125 Ie23 IgG. The composition of I-IV are shown in FIG. 2

important to have the KDEL (SEQ ID NO:9) on the same polypeptide as thetoxin moiety. If no KDEL (SEQ ID NO:9) is present, toxicity is lost. IfKDEL (SEQ ID NO:9) is on the V_(L) domain, cytotoxicity is also lost.The presence of KDEL (SEQ ID NO:9) on V_(L) in addition to V_(H) -toxindoes not change cytotoxic activity. Thus the KDEL (SEQ ID NO:9) sequencemust be on the same polypeptide chain as the toxin.

G) Relative Binding Affinities

Relative binding affinities of the immunotoxins were determined byadding ¹²⁵ I-labeled e23IgG to 10⁵ N87 cells as a tracer with variousconcentrations of the competitor. The binding assays were performed at4° C. for 2 h in RPNH containing 1% bovine serum albumin and 50 mM MES(Sigma) as described (Batra et al. Proc. Natl. Acad. Sci. USA, 89:58678-5871 (1992)). Table 3 shows that there is very little differencein binding affinities among the four molecules. Thus the differences incytotoxicities can be attributed to the location of the KDEL (SEQ IDNO:9) sequence on the toxin molecules.

Example 3 Cytotoxicity and Binding of B3-immunotoxins

Monoclonal antibody B3 is a murine antibody referred to above directedagainst Lewis^(Y) and related carbohydrate antigens which are abundanton the surface of many carcinomas. See Example 1 for a fullerdescription of Lewis^(Y) antigens.

To evaluate the binding affinities and cytotoxic effect on cancer cells,PE with amino acids 1-279 of the amino terminus deleted were modified byinserting variable regions of either B3 heavy or light chains. Theinsertions were made as described above in the Ib domain or at thecarboxyl terminus of domain III. See FIG. 5 for a schematic of the B3immunotoxins.

A) Cytotoxic Activity of B3-immunotoxins Toward B3-antigen ExpressingCell Lines

The cytotoxicity of B3-immunotoxins was determined by measuring thereduction in the incorporation of (³ H)-leucine by A431 cells aftertreatment with immunotoxin Kuan et al. J. Biol Chem., 269: 7610-7616(1994)). A comparison of B1 immunotoxins (see Table 1) indicates thatthe B3-immunotoxins are less cytotoxic than the B1constructs. As Table 4shows, this decrease in cytotoxicity is likely due in part to decreasedbinding affinity.

B) Binding Affinities of B3-immunotoxins

To determine relative binding affinities, increasing concentrations ofeach immunotoxin competed for the binding of (¹²⁵ I)-B3-IgG (or B1-IgGfor comparison) to A431 cells at 4° C. for 2 hours in RPM' containing 1%bovine serum albumin and 50 mM MES as described (Batra et al. Proc.Nat'l. Acad. Sci. USA, 89: 58678 (1992)).

                  TABLE 4                                                         ______________________________________                                        Cytotoxicity and binding of B3-immunotoxins on A431 cells                                      A431      Binding                                            Construct        (IC.sub.50 ng/nM)                                                                       (nM)                                               ______________________________________                                        B3(Fv)-PE38 (LMB7)                                                                             1˜1.5                                                                             550                                                B3(dsFv)PE38     1˜1.5                                                                             25,000                                             B3(VH)-PE35-(VL) 110       >30,000                                            B3(VL-PE35-(VH)  100       6,000                                              B3(VH)-PE33-(VL) 5         30,000                                             B3(VL)-PE33-(VH) 50        5,000                                              B3-IgG                     150                                                ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Cytotoxicity and binding of B3∂B1-immunotoxins on A431           cells                                                                                        A431    Binding                                                               (IC.sub.50 ng/nM)                                                                     (nM)                                                   ______________________________________                                        Protein                                                                       B3(VH)-PE38-(VL) 1˜1.5                                                                             25,000                                             B3VH)-PE35-(VL)  110       >30,000                                            B3(VL)-PE3S-(VH) 100       6,000                                              B3(VH)-PE33-(VL) 5         30,000                                             B3(VL)-PE33-(VH) 50        5,000                                              B3(Fv)-PE38      1˜1.5                                                                             550                                                B3-IgG                     150                                                Construct                                                                     B1(VH)-PE38-(VL) 0.5       2,000                                              B1(VH)-PE33-(VL) 0.25      3,500                                              B1(VH)-PE33      2.0       25,000                                             B1-IgG                     40                                                 ______________________________________                                    

Example 4 Cytotoxicity and Binding of e23-immunotoxins on Cancer Cells

Monoclonal antibody e23 is a murine antibody directed against erbB2antigen. See Example 2 for a fuller description of the erbB2 antigen andthe preparation of e23-immunotoxins.

To evaluate the binding affinities and cytotoxic effect on cancer cells,PE with the first 279 amino acids at the amino terminus deleted weremodified by insertion of variable regions of either e23 heavy or lightchains. The insertions were made in the Ia domain or at the carboxylterminus of domain III.

A) Cytotoxic Activity of e23-immunotoxins Against Cancer Cell Lines

The cytotoxicity of e23-immunotoxins was determined by measuring thereduction in the incorporation of [³ H]-leucine by MCF7 and N-87 celllines after treatment with serial dilutions of the immunotoxins in PBScontaining 0.2% HSA as described previously (Kuan et al. J. Biol Chem.,269: 7610-7616 (1994)). The results are shown in Table 6.

B) Binding Affinities of e23-immunotoxins

To determine relative binding affinities, increasing concentrations ofeach immunotoxin competed for the binding of (¹²⁵ I)-e23-IgG to MCF7 andN-87 cells at 4° C. for 2 hours in RPMI containing 1% bovine serumalbumin and 50 mM MES as described (Batra et al. Proc. Nat'l. Acad. Sci.USA, 89: 58678 (1992)). The results are shown in Table 6.

                  TABLE 6                                                         ______________________________________                                        Cytotoxicity and binding of e23-immunotoxins on cancer cells                             MCF7      Binding  N-87    Binding                                 Construct  (IC50 ng/ml)                                                                            (nM)     (IC.sub.50 ng/ml)                                                                     (nM)                                    ______________________________________                                        e23(VH)PE38-VL                                                                           3.5       110      0.35    120                                     e23VL)PE35-(VH)                                                                          15        65       70      110                                     e23(VL)PE35                                                                              2.2       1,800    42      2,000                                   e23(VH)PE33-(VL)                                                                         30        320      20      210                                     e23(VL)PE33-(VH)                                                                         25        115      3.6     110                                     e23(VL)PE33                                                                              70        5,000    200     >2,000                                  e23-IgG                               4                                       ______________________________________                                    

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 13                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 66 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                                 (B) LOCATION: 1..66                                                 #/note= "forward PCR primer CT119"                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - GGCAACGACG AGGCCGGCGC GGCCAACGGC GGTGGCGGAT CCGAGGTGCA GC - #TGGTGGAA         60                                                                          #           66                                                                - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 51 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                                 (B) LOCATION: 1..51                                                 #/note= "reverse PCR primer CT120"                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #             51TCCGCGC CAGTGGGCTC GGGACCTCCG GAAGCTTTTG C                    - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 54 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - AAACCGAGGC CTTCCGGAGG TGGTGGATCC GAAGTGCAGC TGCAGGAGTC AG - #GA               54                                                                          - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 68 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - TTAGCAGCCG AATTCTTAGA GCTCGTCTTT CGGCGGTTTG CCGGAGGAGA CG - #GTGACCGT         60                                                                          #          68                                                                 - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 54 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - AAACCGAGGC CTTCCGGAGG TGGTGGATCC GAAGTGCAGC TGCAGGAGTC AG - #GA               54                                                                          - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 42 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #  42              TAGG AGACGGTGAC CGTGGTCCCT GC                              - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 68 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - TTAGCAGCCG AATTCTTAGA GCTCGTCTTT CGGCGGTTTG CCGGAGGAGA CG - #GTGACCGT         60                                                                          #          68                                                                 - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Arg Glu Asp Leu Lys                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Lys Asp Glu Leu                                                             - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Ser Gly Gly Gly Gly Ser                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Lys Ala Ser Gly Gly Pro Glu                                                 1               5                                                             - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Gly Gly Gly Gly Ser                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Arg Glu Asp Leu                                                             __________________________________________________________________________

What is claimed is:
 1. A nucleic acid encoding an immunotoxin that doesnot require proteolytic activation for cytotoxic activity, saidimmunotoxin comprising a Pseudomonas exotoxin (PE) having an aminoterminus and a carboxyl terminus, but lacking amino acids 1 through 279,and in which half or more of domain Ib is replaced with part or all ofeither a variable light (V_(L)) chain of an Fv antibody fragment havingan amino terminus and a carboxyl terminus or a variable heavy (V_(H))chain of an Fv antibody fragment having an amino terminus and a carboxylterminus, provided that if only a part of a V_(L) or of a V_(H) chain isused, said part specifically binds to a target molecule.
 2. A nucleicacid of claim 1, wherein half or more of PE domain Ib is replaced withall or part of a variable light chain region.
 3. A nucleic acid of claim1, wherein half or more of PE domain Ib is replaced with all or part ofa variable heavy chain region.
 4. A nucleic acid of claim 1, wherein theFv antibody fragment is of an antibody selected from the groupconsisting of B1, B3, B5, e23, BR96, anti-Tac, RFB4, and HB21.
 5. Thenucleic acid of claim 1, wherein said immunotoxin has KDEL (SEQ ID NO:9)as a carboxyl terminal sequence of said PE.
 6. The nucleic acid of claim1, wherein said amino terminus of said V_(H) region or of said V_(L)region is attached to the PE through a peptide linker.
 7. The nucleicacid of claim 1, wherein said peptide linker is SGGGS (SEQ ID NO:10). 8.The nucleic acid of claim 1, wherein the carboxyl terminus of said V_(H)region or of said V_(L) region is attached to the PE through a peptidelinker.
 9. The nucleic acid of claim 1, wherein said peptide linker isKASGGPE (SEQ ID NO:11).
 10. A method of killing a cell bearing a targetmolecule, said method comprising contacting said cells with animmunotoxin comprising a Pseudomonas exotoxin that does not requireproteolytic activation for cytotoxic activity and wherein half or moreof domain Ib is replaced by part or all of an Fv antibody fragment whichspecifically binds to the target molecule, said Fv fragment having avariable heavy chain region (V_(H)) and a variable light chain region(V_(L)) which V_(H) and V_(L) regions are bound by at least onedisulfide bond, whereby said Pseudomonas exotoxin kills said targetcell.
 11. The method of claim 10 wherein the Fv antibody fragment isfrom an antibody selected from the group consisting of B1, B3, B5, e23,BR96, anti-Tac, RFB4, and HB21.
 12. The method of 10, wherein saidimmunotoxin has KDEL (SEQ ID NO:9) as a carboxyl terminal sequence ofsaid PE.
 13. The method of claim 10, wherein said immunotoxin isselected from the group consisting of B1(dsFv)PE33, B3(VH)-PE33-(VL) andB3(VL)-PE33-(VH).
 14. A method of delivering a Pseudomonas exotoxin (PE)to the cytosol of a cell, said method comprising contacting said cellwith a chimeric molecule, said chimeric molecule comprising a PE thatdoes not require proteolytic cleavage for cytotoxic activity and a heavychain variable region or a light chain variable region of a Fv fragmentof an antibody which specifically binds to the cell, wherein part or allof said heavy chain variable region or said light chain variable regionreplaces half or more of domain Ib of said PE, thereby delivering saidPE to the cytosol of the cell.
 15. The method of claim 14, wherein saidvariable region of an Fv fragment is of a light chain.
 16. The method ofclaim 15, wherein said light chain is bound by a disulfide bond to aheavy chain variable region of an Fv fragment of an antibody.
 17. Themethod of claim 14, wherein said variable region of an Fv fragment is ofa heavy chain.
 18. The method of claim 17, wherein said heavy chain isbound by a disulfide bond to a light chain variable region of an Fvfragment of an antibody.
 19. The method of claim 14, wherein the Fvfragment of an antibody is from an antibody selected from the groupconsisting of B1, B3, B5, c23, BR96, anti-Tac, RFB4, and MB21.
 20. Themethod of claim 14, wherein said chimeric molecule has KDEL (SEQ IDNO:9) as a carboxyl terminal sequence of said PE.
 21. The nucleic acidof claim 14, wherein said amino terminus of said V_(H) region or of saidV_(L) region is attached to the PE through a peptide linker.
 22. Thenucleic acid of claim 21, wherein said peptide linker is SGGGS (SEQ IDNO:10).
 23. The nucleic acid of claim 14, wherein the carboxyl terminusof said V_(H) region or of said V_(L) region is attached to the PEthrough a peptide linker.
 24. The nucleic acid of claim 23, wherein saidpeptide linker is KASGGPE (SEQ ID NO:11).
 25. The method of claim 14,wherein said immnunotoxin is selected from the group consisting ofB1(dsFv)PE33, B3(VL)-PE33-(VH), and B3(V_(H))-PE33-(VL).